Cell display of antibody libraries

ABSTRACT

The present invention relates to a viral vector encoding for a library of antibodies or antibody fragments that are displayed on the cell membrane when expressed in a cell. The present invention provides cells comprising the viral vector nucleic acids and methods of screening the libraries for antibodies or antibody fragments with desired characteristics.

1. CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. No. 60/726,161, filed Oct. 14, 2005, thedisclosure of which is incorporated by reference in its entirety for allpurposes.

2. FIELD OF THE INVENTION

The present invention provides a method for displaying antibodies orantibody fragments on the surface of a cell membrane; a method forproducing a library of cells displaying antibodies or antibody fragmentson the cell surface; cells expressing a library of antibodies orantibody fragments on the cell surface; viral vector libraries forexpressing a library of antibodies or antibody fragments on a cellmembrane; a method of screening for antibodies or antibody fragmentsthat bind to a particular antigen; a method of screening for antibodiesor antibody fragments with improved and/or altered bindingcharacteristics; a method of screening cells expressing and displayingon their cell surface antibodies or antibody fragments that bind to aparticular antigen; a method of screening cells expressing anddisplaying on their cell surface antibodies or antibody fragments withimproved and/or altered binding characteristics; and related kits. Thepresent invention also provides Fc variants with altered ligand binding(e.g., FcγR binding) and/or altered effector function (e.g., ADCCactivity).

3. BACKGROUND OF THE INVENTION

Antibodies are immunological proteins that bind a specific antigen. Inmost mammals, including humans and mice, antibodies are constructed frompaired heavy and light polypeptide chains. Each chain is made up of twodistinct regions, referred to as the variable (Fv) and constant (Fc)regions. The light and heavy chain Fv regions contain the antigenbinding determinants of the molecule and are responsible for binding thetarget antigen. The Fc regions define the class (or isotype) of antibody(IgG for example) and are responsible for binding a number of Fcreceptors and other Fc ligands, imparting an array of importantfunctional capabilities referred to as effector functions. Several keyfeatures of antibodies including but not limited to, specificity fortarget, ability to mediate immune effector mechanisms, and longhalf-life in serum, make antibodies powerful therapeutics. Recombinantscreening methods for isolating antibodies with a desired bindingspecificity have been developed. For example, it is possible to generatelarge expression libraries of binding molecules using combinatorialrecombinant DNA technologies. This is especially true in the field ofantibody engineering, where recombinant antibody libraries routinelycontain more then 10⁹ unique clones. The availability of large librariesof binding molecules has provided a source of binders to most anyligand.

Surface display libraries allow for the enrichment of specific bindingclones by subjecting the organism displaying the binding molecule (e.g.,phage and yeast) to successive rounds of selection (e.g., panning; forreviews see, Trends Biotechnol 9: 408-414; Coomber, et al., 2002,Methods Mol Biol 178: 133-45, Kretzschmar et al., 2002, Curr OpinBiotechol 13: 598-602; Fernandez-Gacio, et al., 2003, Bioorg Med ChemLett. 13:213-216; Lee et al., 2003, Trends Biotechnol 21: 45-52; andKondo, et al., 2004, Appl Microbiol Biotechnol 64: 28-40). Inparticular, advances in phage display antibody libraries have made theman attractive alternative to screening conventional hybridoma-derivedmonoclonal antibodies. Phage display library screening is advantageousover some other screening methods due to the vast number of differentpolypeptides (typically exceeding 10⁹) that can be contained in a singlephage display library. This allows for the screening of a highly diverselibrary in a single screening step.

Display of small peptides or single chain proteins on phage can beadvantageous as long as intracellular processing or post-translationalmodification (of which phage or prokaryotic hosts are not capable) isnot necessary or desired. For example, effective display of aheterologous polypeptide may require various post-translationalmodifications, intracellular structures, and a compliment of specializedenzymes and chaperone proteins that are necessary to transport, toglycosylate, to conform, to assemble, and to anchor the displaypolypeptide properly on the surface of the host cell; however, none ofthese processes can be accomplished by bacteriophage or prokaryotic cellprocesses. Furthermore, prokaryotic cells do not always efficientlyexpress functional eukaryotic proteins.

Bacterial and bacteriophage display systems are also limited by thesmall capacity of the display system, and as such, are more suited forthe display of small peptides as are the recently developed methods forsurface display of small peptides on mammalian cells (see, e.g.,Wolkowicz, et al., 2005, J. Biol. Chem., 280: 15195-15201). As a resultbacteriophage and mammalian antibody display libraries and methodsrequire that only a fragment of an antibody be displayed on the surface.If the purpose is to discover “whole” antibodies then the antibodyfragments must be cloned into a whole antibody. Not only does this addan extra step, but also many antibody fragments have decreased affinityfor an antigen when converted to a whole antibody and such libraries.Furthermore, such methods cannot be used to examine the binding of otherantibody domains such as the Fc region to antibody receptors (e.g., Fcreceptors) or other Fc ligands.

Whole antibody cell surface display systems have been developed for someeukaryotic cells, such as yeast (see, e.g., Boder and Wittrup, 2000,Methods in Enzymology, 328:430-444), but the develop of whole antibodydisplay on mammalian cells lags behind. Furthermore, the size of thelibraries, which can be generated in these systems, is limited. Sincethe chance of isolating antibodies with the desired binding propertiesfrom an antibody library is proportional to the size and diversity ofthe library there is a need for methods to generate large and diverselibraries. This is particularly important if you want to build a naiveantibody library for antibody discovery, for example from un-immunizeddonors. Currently, to build the library size larger than 10⁸ members isa challenge to any eukaryotic cell display technology by usingconventional transfection tools such as transient transfection orelectroporation. Thus, there is a need for antibody cell surface displaylibraries and library screening methods for eukaryotic cells, inparticular mammalian cells, which maintain a large diversity, buteliminate any of the issues discussed supra. Such a system would beparticularly useful for the identification of antibody variants inregions outside of the variable domain such as in the Fc region.Modifications, including amino acid deletions, substitutions andadditions, of the Fc region have been demonstrated to alter the bindingof the Fc region to its ligands and/or receptors resulting in aconcomitant change in effector function (see, e.g., (Shields et al.,2001, J Biol Chem 276:6591-6604 and Presta et al., 2002, Biochem SocTrans 30:487-490 and U.S. Patent Publication 2004/0132101). Thus, bymodifying the Fc region the therapeutic effectiveness of Fc containingmolecules can be improved. A system for whole antibody cell surfacedisplay on mammalian cells would facilitate the rapid identification ofantibodies with modified Fc regions having altered effector function.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

4. SUMMARY OF THE INVENTION

The present invention relates to a recombinant antibody or fragmentthereof that is displayed on the extracellular surface of the cellmembrane, referred to herein as an “antibody of the invention” and liketerms. In certain embodiments, a recombinant antibody of the inventioncomprises a heavy chain or a fragment thereof and optionally a lightchain or a fragment thereof, wherein either the heavy chain or lightchain further comprises an amino acid sequence that targets the antibodyor fragment thereof to the cell surface. In one embodiment, arecombinant antibody of the invention comprises a full length heavychain having an amino acid sequence that targets the antibody to thecell surface wherein said amino acid sequence is fused to the C terminusof said heavy chain and may further comprise a full length light chain.In still another embodiment, a recombinant antibody of the inventioncomprises a portion of a heavy chain having an amino acid sequence thattargets the antibody to the cell surface wherein said amino acidsequence is fused to the C terminus of said heavy chain portion and mayfurther comprise a light chain or fragment thereof. In a specificembodiment, said amino acid sequence that targets the antibody to thecell surface is a transmembrane domain. In another embodiment, saidamino acid sequence that targets the antibody to the cell surface is aGPI anchor signal sequence.

The present invention further relates to vectors comprisingpolynucleotides encoding a recombinant antibody or fragment thereof thatis displayed on the extracellular surface of the cell membrane, referredto herein as a “vector of the invention”. In one embodiment, a vector ofthe invention is a viral vector. In a specific embodiment, a vector ofthe invention is an adenoviral vector.

The present invention also relates to libraries comprising recombinantantibodies or fragments thereof that are displayed on the extracellularsurface of the cell membrane, referred to herein as a “library of theinvention”. In one embodiment, a library of the invention comprises alibrary of heavy chain variable regions; it may further comprise alibrary of light chain variable regions; and it may further comprise alibrary of variant Fc regions. In one embodiment, a library of thepresent invention is a library of cells comprising polynucleotidesencoding a recombinant antibody or fragment thereof that is displayed onthe extracellular surface of the cell membrane.

The invention also provides methods of screening a library of theinvention comprising recombinant antibodies or fragments thereof thatare displayed on the extracellular surface of the cell membrane. In oneembodiment, a method of screening a library allows the identification ofan antibody or fragment thereof that binds a specific antigen. In oneembodiment, a method of screening a library allows the identification ofan antibody or fragment thereof having an altered binding to a specificantigen. In one embodiment, a method of screening a library allows theidentification of an antibody or fragment having an altered binding toeffector molecules (e.g., FcγRs and/or C1q).

The present invention provides variant Fc regions having altered bindingto effector molecules (e.g., FcγRs and/or C1q). The present inventionalso provides variant Fc regions having an altered effector function. Inone embodiment, a variant Fc region of the invention has a reducedantibody dependent cell-mediated cytotoxicity (ADCC).

5. BRIEF DESCRIPTION OF THE FIGURES

For the purpose of illustrating exemplary embodiments of the invention,drawings are provided herein.

FIG. 1A-C. Schematic representation of non-limiting examples ofexpression cassettes (I-VI) that may be used for the cell surfacedisplay of an antibody or a fragment thereof.

FIG. 2. Fluorescence intensity profile of stained HEK-293T cellsexpressing an antibody fusion polypeptide comprising a transmembranedomain or a GPI anchor signal. HEK-293T cells expressing an anti-EphA2antibody comprising a heavy chain fused to i) the GPI anchor signal ofcarboxypeptidase M (CM GPI), ii) a frameshift mutant GPI anchoringsignal of DAF (DAF mutGPI), iii) a variant GPI anchoring signal of DAF(DAF vGPI), or iv) the transmembrane domain of thrombomodulin (TM; twoindependently isolated clones analyzed) were stained with either FITCconjugated anti-human IgG or biotinylated EphA2-Fc fusion protein/FITCconjugated anti-biotin antibody and analyzed with a flow cytometer.Similarly stained (293T+FITC), as well as non-stained (293T), HEK-293Tcells were included as negative control. Cells expressing CM GPI, DAFvGPI, or TM fused anti-EphA2 antibody displayed fluorescence intensitythat was significantly higher than that of the control cells. Cellsexpressing a DAF mutGPI fused anti-EphA2 antibody displayed fluorescenceintensity that was substantially the same as that of the controlHEK-293T cells

FIG. 3. Fluorescence intensity profile of affinity stained HEK-293Tcells transfected with different amounts of plasmid DNA encoding ananti-EphA2 fusion antibody comprising a DAF vGPI. HEK-293T cells weretransfected with different amounts (0.05, 0.1, 0.5, 1.0, 2.0, 4, and 10μg) of plasmid DNA. Transfected cells were first contacted with anFcγRIIIA-streptavidin fusion protein and then stained with FITCconjugated anti-streptavidin antibody. The cells were subsequentlyanalyzed on a flow cytometer. Non-transfected HEK-293T cells wereincluded as negative control. The flow cytometry profiles of all thetransfected cell populations show a significant shift in meanfluorescence intensity compared to the control cells.

FIG. 4. Fluorescence intensity profile of affinity stained HEK-293Tcells expressing an anti-EphA2 fusion antibody comprising a DAF vGPI.HEK-293T cells were transfected with 10 μg of plasmid DNA encoding ananti-EphA2 fusion antibody comprising a DAF vGPI. Transfected cells weredivided into aliquots and incubated with different dilutions ofFcγRIIIA-streptavidin fusion protein (1:500, 1:1000, 1:2000, 1:3000,1:4000 and 1:5000). Cells were then stained with FITC conjugatedanti-streptavidin antibody and analyzed by flow cytometry. Flowcytometry profiles show that the use of decreasing amounts ofFcγRIIIA-streptavidin fusion protein resulted in decreasing stainingintensity.

FIG. 5. Sort parameters used for the isolation of Fc variants with lowbinding affinity for FcγRIIIA. HEK-293 cells were transientlytransfected with an Fc variant Insertion Library (Fc-IL) and stainedwith FcγRIIIA-streptavidin fusion protein/FITC conjugatedanti-streptavidin antibody. (A) Cells with low fluorescence intensity(approximately 10% of total according to M1 marker) were isolated usinggate R2. (B) The isolated cell population displayed uniform lowfluorescence intensity.

FIG. 6. Fluorescence intensity profile of stained HEK-293 cellsexpressing Fc variants of an anti-EphA2 fusion antibody comprising a DAFvGPI. HEK-293 cells expressing a wild type (A and D), K246E Fc variant(B and E) or InR236/237 Fc variant (C and F) of an anti-EphA2 fusionantibody comprising a DAF vGPI were analyzed by flow cytometry. Cellswere stained either with FITC conjugated anti-human IgG antibody (A-C)or with FcγRIIIA-streptavidin fusion protein/FITC conjugatedanti-streptavidin antibody. Each panel contains the flow cytometryprofile of antibody expressing (black line) and control (grey line)HEK-293 cells. All three cell populations stained with FITC conjugatedanti-human IgG antibody showed similar levels of fluorescence intensitythat was significantly different from that of observed for the controlcells. When stained with FcγRIIIA-streptavidin fusion protein/FITCconjugated anti-streptavidin antibody, only cells expressing wild typeor K246E Fc variant antibodies displayed fluorescence intensity that wassignificantly higher than that of observed for the control HEK-293cells.

FIG. 7. ELISA based FcγRIIIA binding curve of Fc variants InR236/237,InN238/239, and InV238/239. Binding curves for FcγRIIIA interaction withwild type or Fc variants InR236/237, InN238/239, and InV238/239anti-EphA2 antibodies were established using standard ELISA protocols.An IgG4 isotype antibody of the same antigen specificity was included asa negative control. The binding curves show that interaction betweenFcγRIIIA and the InR236/237, InN238/239, or InV238/239 Fc variant isweaker than that of between FcγRIIIA and the Fc region of the IgG4negative control antibody. FcγRIIIA displayed robust binding to thepositive control antibody comprising a wild type Fc region.

FIG. 8. ELISA based C1q binding curve of Fc variants InG240/241,InR234/235, InL238/239, InE238/239, InS239/240, InR237/238, andK246R/L251E/T260R. Binding curves for C1q interaction with wild type orFc variants InG240/241, InR234/235, InL238/239, InE238/239, InS239/240,InR237/238, and K246R/L251E/T260R anti-EphA2 antibodies were establishedusing standard ELISA protocols. An IgG4 isotype antibody of the sameantigen specificity was included as a negative control. Each of the Fcvariants shows reduced binding to C1q as compared to the wild typeantibody.

FIG. 9. Percent binding of Fc variants K246R/L251E/T260R, InR236/237,InN238/239, InV238/239, InG240/241, InR234/235, InL238/239, InE238/239,InS239/240 and InR237/238 to THP-1 cells. THP-1 monocytes expressingFcγRI and FcγRII, but not FcγRIIIA, were contacted with wild type or Fcvariants K246R/L25 1 E/T260R, InR236/237, InN238/239, InV238/239,InG240/241, InR234/235, InL238/239, InE238/239, InS239/240 andInR237/238 anti-EphA2 antibodies. The percentage of THP-1 monocytesbound by each antibody was determined by staining the cells with FITCconjugated anti-human IgG Fab and analyzing them on a flow cytometer.The obtained results show reduced binding of FcγRI and FcγRII by each ofthe Fc variants tested compared to the wild type antibody.

FIG. 10. Percent binding of Fc variants InR236/237, InN238/239, andInV238/239 to NK cells. NK cells expressing FcγRIIIA were contacted withwild type or Fc variants InR236/237, InN238/239, and InV238/239anti-EphA2 antibodies. The percentage of NK cells bound by each antibodywas determined by staining the cells with FITC conjugated anti-human IgGFab and analyzing them on a flow cytometer. An IgG4 isotype antibody ofthe same antigen specificity was included as a negative control. Theobtained results show reduced binding of FcγRIIIA by each of the Fcvariants tested compared to the wild type antibody.

FIG. 11. ELISA based EphA2 binding curve of Fc variants InR236/237,InN238/239, InV235/236, and InG238/239. Human EphA2 binding of wild typeand Fc variants InR236/237, InN238/239, InV235/236, and InG238/239anti-EphA2 antibodies was determined using standard ELISA protocols.Vitaxin® (anti-α_(V)β₃ integrin antibody) and an anti-HMGB1 antibodywere included in the assay as negative controls. The results show thateach of the Fc variants tested binds to human Eph-A2 with an affinitysimilar to that of the wild type antibody.

FIG. 12. In vitro ADCC activity of Fc variants InR236/237, InN238/239,InV238/239. ADCC activity of Fc variants InR236/237, InN238/239,InV238/239 was determined at 50:1 (left panel) or a 25:1 (right panel)effector to target cell ratios using standard protocols. Wild typeanti-EphA2 antibody and an anti-CD4 antibody (R347) were included aspositive and negative controls, respectively. EphA2 expressing A549cells were used as targets. Purified human peripheral blood mononuclearcells were used as effectors. Cytotoxicity was determined at antibodyconcentrations between 0.1 and 10000 ng/ml. Each of the Fc variantstested shows ADCC activity similar to that of the negative controlantibody. The wild type anti-EphA2 antibody displayed robust ADCCactivity under the same conditions.

FIG. 13. Flow cytometry profiles from Proof of Principle SelectionExperiment I. Cells were incubated with αVβ3-biotin followed by stainingwith FITC conjugated anti-human IgG-Fc and APC-conjugated streptavidin.Samples displayed are as follows: (A) negative control 293A cells, (B)293A cells infected with a cell surface displayed 3F2 anti-EphA2antibody encoding ts369 mutant adenovirus, (C) 293A cells infected witha cell surface displayed Abegrin anti-αVβ3 integrin ScFvFc encodingts369 mutant adenovirus, (D) 293A cells infected with the artificiallibrary before magnetic bead mediated selection, (E) 293A cells infectedwith the artificial library after magnetic bead mediated selection. GateP6 was used to sort double positive cells.

FIG. 14. Flow cytometry profiles from Proof of Principle SelectionExperiment II. Cells were incubated with biotinylated EphA2 followed bystaining with FITC conjugated anti-human IgG-Fc and APC-conjugatedstreptavidin. Samples displayed are as follows: (A) negative control293A cells, (B) 293A cells infected with a cell surface displayedAbegrin anti-αVβ3 integrin antibody encoding ts369 mutant adenovirus,(C) 293A cells infected with a cell surface displayed 3F2 anti-EphA2ScFvFc encoding ts369 mutant adenovirus, (D) 293A cells infected withthe artificial library before magnetic bead mediated selection, (E) 293Acells infected with the artificial library after magnetic bead mediatedselection. Gate P6 was used to sort double positive cells. Percentage ofcells covered by Gate P6 is displayed in the panels.

FIG. 15. Flow cytometry profiles from Proof of Principle SelectionExperiment III. Cells were incubated with biotinylated EphA2 followed bystaining with FITC conjugated anti-human IgG-Fc and APC-conjugatedstreptavidin. Samples displayed are as follows: (A) negative control293A cells, (B) 293A cells infected with a cell surface displayedanti-PCDGF antibody encoding ts369 mutant adenovirus, (C) 293A cellsinfected with a cell surface displayed 10C12 anti-EphA2 ScFvFc encodingts369 mutant adenovirus, (D) 293A cells infected with the artificiallibrary before magnetic bead mediated selection, (E) 293A cells infectedwith the artificial library after magnetic bead mediated selection. GateP6 was used to sort double positive cells. Percentage of cells coveredby Gate P6 is displayed in the panels.

6. DEFINITIONS

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, camelised antibodies, chimeric antibodies,single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab′) fragments, and anti-idiotypic (anti-Id) antibodies (including,e.g., anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. In particular, antibodiesinclude immunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site, these fragments may or may not be fused to anotherimmunoglobulin domain including but not limited to, an Fc region orfragment thereof. As used herein, the terms “antibody” and “antibodies”also include the Fc variants, full-length antibodies and Fcvariant-fusions comprising Fc regions, or fragments thereof. Fcvariant-fusions include but are not limited to, scFv-Fc fusions,variable region (e.g., VL and VH)—Fc fusions, scFv-scFv-Fc fusions.Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

The complementarity determining regions (CDRs) residue numbers referredto herein are those of Kabat et al. (1991, NIH Publication 91-3242,National Technical Information Service, Springfield, Va.). Specifically,residues 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) in the light chainvariable domain and 31-35 (CDR1), 50-65 (CDR2) and 95-102 (CDR3) in theheavy chain variable domain. Note that CDRs vary considerably fromantibody to antibody (and by definition will not exhibit homology withthe Kabat consensus sequences). Maximal alignment of framework residuesfrequently requires the insertion of “spacer” residues in the numberingsystem, to be used for the Fv region. It will be understood that theCDRs referred to herein are those of Kabat et al. supra. In addition,the identity of certain individual residues at any given Kabat sitenumber may vary from antibody chain to antibody chain due tointerspecies or allelic divergence.

As used herein “Fc region” includes the polypeptides comprising theconstant region of an antibody excluding the first constant regionimmunoglobulin domain. Thus Fc refers to the last two constant regionimmunoglobulin domains of IgA, IgD, and IgG, and the last three constantregion immunoglobulin domains of IgE and IgM, and the flexible hingeN-terminal to these domains. For IgA and IgM Fc may include the J chain.For IgG, Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2and Cγ3) and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Althoughthe boundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to comprise residues C226 or P230 to itscarboxyl-terminus, wherein the numbering is according to the EU index asin Kabat et al. (1991, NIH Publication 91-3242, National TechnicalInformation Service, Springfield, Va.). The “EU index as set forth inKabat” refers to the residue numbering of the human IgG1 EU antibody asdescribed in Kabat et al. supra. Fc may refer to this region inisolation, or this region in the context of an antibody, antibodyfragment, or Fc fusion protein. An Fc variant protein may be anantibody, Fc fusion, or any protein or protein domain that comprises anFc region. Particularly preferred are proteins comprising variant Fcregions, which are non-naturally occurring variants of an Fc region. Theamino acid sequence of a non-naturally occurring Fc region (alsoreferred to herein as a “variant Fc region”) comprises a substitution,insertion and/or deletion of at least one amino acid residue compared tothe wild type amino acid sequence. Any new amino acid residue appearingin the sequence of a variant Fc region as a result of an insertion orsubstitution may be referred to as a non-naturally occurring amino acidresidue. Note: Polymorphisms have been observed at a number of Fcpositions, including but not limited to Kabat 270, 272, 312, 315, 356,and 358, and thus slight differences between the presented sequence andsequences in the prior art may exist.

As used herein, the term “transmembrane domain” refers to the domain ofa peptide, polypeptide or protein that is capable of spanning the plasmamembrane of a cell. These domains can be used to anchor an antibody onthe cell membrane.

A “chimeric antibody” is a molecule in which different portions of theantibody are derived from different immunoglobulin molecules such asantibodies having a variable region derived from a non-human antibodyand a human immunoglobulin constant region. Methods for producingchimeric antibodies are known in the art. See e.g., Morrison, 1985,Science 229:1202; Oi et al., 1986, BioTechniques 4:214; Gillies et al.,1989, J. Immunol. Methods 125:191-202; and U.S. Pat. Nos. 5,807,715,4,816,567, and 4,816,397, CDR-grafting (EP 239,400; PCT Publication No.WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089),veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991,Molecular Immunology 28(4/5): 489-498; Studnicka et al., 1994, ProteinEngineering 7:805; and Roguska et al., 1994, PNAS 91:969), and chainshuffling (U.S. Pat. No. 5,565,332).

A “humanized antibody” is an antibody or its variant or fragment thereofwhich is capable of binding to a predetermined antigen and whichcomprises a framework region having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains (Fab, Fab′, F(ab′)2, Fabc, Fv) in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin(i.e., donor antibody) and all or substantially all of the frameworkregions are those of a human immunoglobulin consensus sequence. In oneembodiment, a humanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Ordinarily, the antibody will contain both the lightchain as well as at least the variable domain of a heavy chain. Theantibody also may include the CH1, hinge, CH2, CH3, and CH4 regions ofthe heavy chain. The humanized antibody can be selected from any classof immunoglobulins, including, but not limited to, IgM, IgG, IgD, IgAand IgE, and any isotype, including, but not limited to, IgG1, IgG2,IgG3 and IgG4. In another embodiment, the constant domain is acomplement fixing constant domain where it is desired that the humanizedantibody exhibit cytotoxic activity, and the class is typically IgG1.Where such cytotoxic activity is not desirable, the constant domain maybe of the IgG 2 class. The humanized antibody may comprise sequencesfrom more than one class or isotype, and selecting particular constantdomains to optimize desired effector functions is within the ordinaryskill in the art. The framework and CDR regions of a humanized antibodyneed not correspond precisely to the parental sequences, e.g., the donorCDR or the consensus framework may be mutagenized by substitution,insertion or deletion of at least one residue so that the CDR orframework residue at that site does not correspond to either theconsensus or the import antibody. Such mutations, however, will not beextensive. In one embodiment, at least 75%, at least 90%, and or atleast 95% of the humanized antibody residues will correspond to those ofthe parental framework region (FR) and CDR sequences. Humanized antibodycan be produced using variety of techniques known in the art, includingbut not limited to, CDR-grafting (European Patent No. EP 239,400; PCTPublication No. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101,and 5,585,089), veneering or resurfacing (European Patent Nos. EP592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5):489-498; Studnicka et al., 1994, Protein Engineering 7(6): 805-814; andRoguska et al., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No.5,565,332), and techniques disclosed in, e.g., U.S. Pat. No. 6,407,213,U.S. Pat. No. 5,766,886, PCT Patent Publication WO 93/17105, Tan et al.,2002, J. Immunol. 169:1119-25, Caldas et al., 2000, Protein Eng. 13:353-60, Morea et al., 2000, Methods 20: 267-79, Baca et al., 1997, J.Biol. Chem. 272: 10678-84, Roguska et al., 1996, Protein Eng. 9:895-904, Couto et al., 1995, Cancer Res. 55 (23 Supp): 5973s-5977s,Couto et al., 1995, Cancer Res. 55: 1717-22, Sandhu J S, 1994, Gene 150:409-10, and Pedersen et al., 1994, J. Mol. Biol. 235: 959-73). Often,framework residues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter and/orimprove antigen binding. These framework substitutions are identified bymethods well known in the art, e.g., by modeling of the interactions ofthe CDR and framework residues to identify framework residues importantfor antigen binding and sequence comparison to identify unusualframework residues at particular positions. (See, e.g., Queen et al.,U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature 332:323).

As used herein the term “multiplicity of infection” (MOI) means thenumber of infectious virus particles per cell.

The term “ADCC” (antibody-dependent cell-mediated cytotoxicity) refersto a cell-mediated reaction in which nonspecific cytotoxic cells thatexpress Fc receptors (FcR) (e.g. natural killer (NK) cells, neutrophils,and macrophages) recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. The primary cells formediating ADCC, NK cells, express FcγRIII only, whereas monocytesexpress FcγRI, FcγRII and FcγRIII. To assess ADCC activity of a moleculeof interest, an in vitro ADCC assay (e.g. such as that described in U.S.Pat. No. 5,500,362 and U.S. Pat. No. 5,821,337) may be performed. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and natural killer (NK) cells.

“Complement dependent cytotoxicity” and “CDC” refer to the lysing of atarget cell in the presence of complement. The complement activationpathway is initiated by the binding of the first component of thecomplement system (C1q) to a molecule, an antibody for example,complexed with a cognate antigen. To assess complement activation, a CDCassay, e.g. as described in Gazzano-Santoro et al., 1996, J. Immunol.Methods, 202:163, may be performed.

7. DETAILED DESCRIPTION

The present invention relates to a recombinant antibody or fragmentthereof that is displayed on the extracellular surface of the cellmembrane, referred to herein as an “antibody of the invention” and liketerms.

In one embodiment, an antibody of the invention is a murine antibody, achimeric antibody, a humanized antibody or human antibody. In oneembodiment, an antibody of the invention is a human antibody.

In one embodiment, an antibody of the invention is of an immunoglobulintype selected from the group consisting of IgA, IgE, IgM, IgD, IgY andIgG.

In one embodiment, a recombinant antibody of the invention comprises aheavy chain or fragment thereof having an amino acid sequence thattargets the antibody to the cell surface. In one embodiment, arecombinant antibody of the invention comprises a heavy chain orfragment thereof having an amino acid sequence that targets the antibodyto the cell surface fused to the C terminal end of said heavy chain orfragment thereof.

In another embodiment, a recombinant antibody of the invention comprisesa light chain or fragment thereof having an amino acid sequence thattargets the antibody to the cell surface. In a specific embodiment, arecombinant antibody of the invention comprises a light chain orfragment thereof having an amino acid sequence that targets the antibodyto the cell surface fused to the C terminal end of said light chain orfragment thereof.

In one embodiment, a recombinant antibody of the invention comprises afull length heavy chain having an amino acid sequence that targets theantibody to the cell surface wherein said amino acid sequence is fusedto the C terminus of said heavy chain and may further comprise a fulllength light chain or a fragment thereof. In still another embodiment, arecombinant antibody of the invention comprises a portion of a heavychain having an amino acid sequence that targets the antibody to thecell surface wherein said amino acid sequence is fused to the C terminusof said heavy chain portion and may further comprise a light chain orfragment thereof.

In a specific embodiment, said amino acid sequence that targets theantibody to the cell surface is a transmembrane domain. In anotherembodiment, said amino acid sequence that targets the antibody to thecell surface is a GPI anchor signal sequence.

The present invention further relates to vectors comprisingpolynucleotides encoding a recombinant antibody or fragment thereof thatis displayed on the extracellular surface of the cell membrane, referredto herein as a “vector of the invention”. In one embodiment, a vector ofthe invention is capable of replication. In one embodiment, a vector ofthe invention is a viral vector. In one embodiment, a vector of theinvention is an adenoviral vector, a baculoviral vector, an adenoassociated viral vector, a herpes viral vector or a lentiviral vector.In a specific embodiment, a vector of the invention is an adenoviralvector.

The present invention also relates to libraries comprising recombinantantibodies or fragments thereof that are displayed on the extracellularsurface of the cell membrane, referred to herein as a “library of theinvention”. The present invention provides antibody or antibody fragmentlibraries and methods of screening cells displaying the library ofantibodies and/or antibody fragment on the cell surface. These methodsinvolve using vectors including, but not limited to, viral vectors todeliver an antibody library to cells, wherein expression of the libraryresults in the display of the antibodies on the cell surface. Also,provided are viral vectors and cells encoding and/or displaying anantibody and/or antibody fragment library.

In one embodiment, a library of the invention may comprise a library ofheavy chain variable regions; it may further comprise a library of lightchain variable regions; and it may further comprise a library of variantFc regions.

In one embodiment, a library of the present invention is a library ofcells comprising polynucleotides encoding a recombinant antibody orfragment thereof that is displayed on the extracellular surface of thecell membrane. In one embodiment, mammalian cells comprise a library ofthe invention. In a specific embodiment, a library of the invention iscomprised by cells selected from the group consisting of NSO cells, CHOcells, Vero cells, Sf-9 cells, COS7 cells, and 293 cells.

The invention also provides methods of screening a library of theinvention comprising recombinant antibodies or fragments thereof thatare displayed on the extracellular surface of the cell membrane. In oneembodiment, a method of screening a library allows the identification ofan antibody or fragment thereof that binds a specific antigen. In oneembodiment, a method of screening a library allows the identification ofan antibody or fragment thereof having an altered binding for a specificantigen. In one embodiment, a method of screening a library allows theidentification of an antibody or fragment having an altered binding foreffector molecules (e.g., FcγRs and/or C1q).

The present invention provides variant Fc regions having altered bindingto effector molecules(e.g., FcγRs and/or C1q). The present inventionalso provides variant Fc regions having an altered effector function. Inone embodiment, a variant Fc region of the invention has a reducedantibody dependent cell-mediated cytotoxicity (ADCC).

The present invention provides a method for selecting mammalian cellsthat express at least one antibody or a fragment thereof with desirablebinding characteristics wherein said method comprises: a) introductioninto mammalian cells a library of nucleic acids in an expression vector,wherein said nucleic acid encodes an antibody, or an antibody fragmentthat is displayed on the cell surface; b) culturing the mammalian cellscomprising the library to allow expression and cell surface presentationof each vector encoded polypeptides; c) contacting the cells with anantigen; and d) isolating the cells that comprise at least one cellsurface displayed polypeptide that binds to the antigen. The presentinvention further provides 1) recovering nucleic acids from the isolatedmammalian cells; 2) amplifying nucleic acids encoding at least oneantibody variable region from the nucleic acids; 3) inserting theamplified nucleic acids into a second vector, wherein the second vector,with the inserted nucleic acids, encodes a secreted soluble antibody and4) transforming a host cell with the second vector.

In certain embodiments the antibody or fragment thereof comprises anamino acid sequence that can target the polypeptide for cell surfacedisplay (examples include, but are not limited to, transmembrane domainsequences and GPI anchor signal sequences). In one embodiment, the heavychain of the antibody or fragment thereof comprises the amino acidsequence that can target the polypeptide for cell surface display. Inanother embodiment, the light chain of the antibody or fragment thereofcomprises the amino acid sequence that can target the polypeptide forcell surface display.

In one embodiment, most or all cells in a library of cells express onlyone clonal antibody. In another embodiment, most or all cells in alibrary of cells express at least two different antibodies. In oneembodiment, a vector of the invention codes for an antibody heavy chainor fragment thereof. In another embodiment, a vector of the inventionadditionally codes for an antibody or antibody fragment light chain,wherein both the heavy chain and light chain are expressed in transducedcells. In certain embodiments, a vector of the invention is a viralvector.

The invention further provides viral vectors encoding a library ofantibodies or antibody fragments that are displayed on the cell membranewhen expressed in a cell. The present invention provides cellscomprising a viral vector nucleic acid and methods of screening thelibraries for antibodies or antibody fragments with desiredcharacteristics.

The present invention also provides methods for screening antibodiesbased on antibody dependent cell-mediated cytotoxicity (ADCC) effect. Inone embodiment, a viral vector encodes a library of antibody Fcvariants.

7.1 Viral Vectors

Viral vectors of the present invention comprise nucleic acids that codefor a library of heavy chain sequences for an antibody or antibodyfragment that when expressed in a cell, the antibody or antibodyfragment is bound to the cell surface. In one embodiment, when bound tothe cell surface, the library of antibodies or antibody fragments is onthe extracellular side of the cell membrane. In one embodiment, theviral vector library further encodes a library of light chain sequencesof antibodies or antibody fragments (See examples I-VI in FIG. 1A-C).

Viral vectors offer several advantages for delivering an antibody orantibody fragment library. The diversity of the library is oneconsideration when preparing and screening the antibody library. Phagedisplay antibody libraries can typically have a diversity of about 109unique antibodies. In the case of cell display antibody libraries, ifeach cell expresses only one unique antibody one would need to utilizeat least 10⁹ and generally 10¹⁰ or more cells to achieve an equivalentdiversity. This is a large number of cells to handle for screening andit would be preferable to work with a smaller number of cells, whilemaintaining diversity. In the present invention, viral vectors areutilized to deliver the antibody or antibody fragment libraries. Thisallows one to infect at chosen multiplicities of infection (MOI). TheMOI can be adjusted to display a certain average number of uniqueantibodies per cell. For example, an MOI of 50 may be used which willallow the expression of an average of 50 unique antibodies per cell. Aswith other library screening methods, the methods of the presentinvention may include a first round of screening/enrichment. In someembodiments of the invention, the cells are infected with an MOI of atmost: 10⁻², 10⁻¹, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 50, 100, 200,500, 10³, 10⁴, or 10⁵. Optionally, further rounds of screening may beperformed by screening the viral vectors coding for the selectedantibodies. In one embodiment, the secondary rounds of selection utilizea lower MOI than in the first round of selection.

Any viral vector may be utilized in accordance with the presentinvention. Viral vectors include, but are not limited to, retroviralvectors, vaccinia vectors, lentiviral vectors, herpes virus vectors(e.g., HSV), baculoviral vectors, cytomegalovirus (CMV) vectors,papillomavirus vectors, simian virus (SV40) vectors, Sindbis vectors,semliki forest virus vectors, adenoviral vectors, and adeno-associatedviral (AAV) vectors. In one embodiment, the viral vector is a high titerviral vector. A high titer viral vector is one that can be isolatedand/or concentrated to a titer of at least 10⁹ viral particles permilliliter. In one embodiment, the viral vector antibody library is ahigh titer viral vector. In one embodiment, the viral vector of theinvention is an adenovirus, baculovirus, AAV or herpes virus vector. Inone embodiment, the viral vector is not a vaccinia vector.

In one embodiment, the viral vector is an AAV vector. In one embodiment,the AAV vectors encode both an antibody heavy and light chain library.For examples and further description of AAV vectors encoding antibodiessee US2005003482, US20040265955 and Fang et al. Nat Biotechnol. 2005May; 23(5):584-90. These vectors can be utilized in the presentinvention by cloning a GPI anchor or transmembrane coding sequencein-frame with the antibody heavy chain as described herein.

In one embodiment, the viral vector is an adenoviral vector. Anadenoviral vector may be any vector derived from an adenovirus whereinthe nucleic acid of the viral vector can be packaged into adenoviralcapsid proteins. The adenoviral vector may be derived from anyadenoviral serotype or may even be a chimeric adenoviral vector withdifferent portions derived from at least 2 different adenoviralserotypes. The adenoviral vector may be derived from a human or anotheranimal adenovirus. In one embodiment, the adenoviral vector is derivedfrom human adenovirus serotype 2, 3, 5, 12, 35 or 40. The adenoviralvectors may be replication competent or incompetent in relation to thecell being infected. In one embodiment, the adenoviral vector isreplication competent in relation to the cell being infected. Forexample, the adenoviral vector may comprise a deletion of the E1 regionand this vector is used to infect a 293 cell that expresses the E1 genesin trans, thus allowing the adenoviral vector to replicate. Many otheradenoviral vector gene deletions and corresponding complementing cellline combinations are known in the art. In one embodiment, theadenoviral vector may be replication incompetent in relation to the cellbeing infected. For example, an E1 deleted adenoviral vector used toinfect A549 cell. In one embodiment, the replication incompetentadenoviral vector is rescued with a helper virus (e.g., expressing E1proteins). In one embodiment, 1) cells are infected with a replicationincompetent adenoviral virus coding for an antibody that will bedisplayed on the cell surface; 2) cells are screened and sorted forthose displaying antibodies with desired binding properties; 3)infecting the positive sorted cells with a helper virus to rescue theviral vectors. The helper virus can be any virus that expresses at leastone complementing gene product for the adenoviral vector (e.g. E1proteins for an E1 deleted adenovirus). In one embodiment, the helpervirus is an adenovirus. In one embodiment, the adenoviral vector may bereplication incompetent as a result of harboring a temperature sensitivemutation. For example, an adenoviral vector comprising the ts369mutation (Hasson, T. B. et al., J Virol 63(9):3612-21 (1989)) isreplication compromised when cultured at 40° C. but can be rescued byculturing at lower temperatures. In one embodiment, 1) cells areinfected at the non-permissive temperature with a temperature sensitiveadenoviral vector coding for an antibody that will be displayed on thecell surface; 2) cells displaying antibodies with desired bindingproperties are selected; 3) virus is rescued from the selected cells byculturing said cells at a temperature permissive for viral replication.

In one embodiment, the viral vector encodes the heavy chain of anantibody or a fragment thereof In another embodiment, the viral vectoradditionally encodes a light chain of an antibody or a fragment thereof,and is therefore capable of expressing, both heavy and light chainantibody polypeptides. In this embodiment, both the heavy and lightchains are displayed together on the cell surface. In one embodiment,the heavy and light chains are displayed as a whole antibody on the cellsurface. In another embodiment, the heavy and light chains are displayedas an antibody fragment on the cell surface. In one embodiment, thelight chain is placed 5′ (upstream) of the heavy chain coding sequence.Not wishing to be bound by theory, this may avoid an excess of toxicfree heavy chain (Proudfoot, 1986, Nature, 322:562-565; and Kohler,1980, Proc. Natl. Acad. Sci. USA, 77:2 197). The coding sequences forthe heavy and light chains may comprise cDNA or genomic DNA.

In one embodiment, the nucleic acid of the viral vector furthercomprises a coding region for a selectable marker. A number of selectionsystems/markers may be used, including but not limited to, the herpessimplex virus thyinidine kinase (Wigler et al, Cell 11:223 (1977)),hypoxanthineguanine phosphoribosyltransferase (Szybalski & Szybalski,Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al, Cell 22:8 17 (1980)) genes can beemployed in tk-, hgprt- or aprt-cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al, Natl. Acad. Sci. USA 77:357 (1980); OHare et al, Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418(clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62: 191-217 (1993); TIB TECH 11 :155-215 (May 1993)); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Sambrook et al., (eds.),Molecular cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, NY, (2001); Ausubel et al. (eds.), Current Protocols in MolecularBiology, John Wiley & Sons, NY (1998); Kriegler, Gene Transfer andExpression, A Laboratory Manual, Stockton Press, NY (1990); and inChapters 12 and 13, Dracopoli et al. (eds), Current Protocols in HumanGenetics, John Wiley & Sons, NY (1999); Colberre-Garapin et al, 1981, J.Mol. Biol. 150:1.

As described herein, in one embodiment, the whole recombinant antibodymolecule, is expressed. In another embodiment, fragments (e.g., Fabfragments, F(ab′) fragments, and epitope-binding fragments) of theimmunoglobulin molecule are expressed.

7.2 Nucleic Acids

When the library of sequences coding for an antibody or antibodyfragment is expressed, the antibody or antibody fragment is the bound tothe cell surface. To accomplish this the nucleic acids of the viralvector library comprises at least two operatively linked coding regionssee, e.g., FIG. 1. The first coding region codes for the an antibodychain (e.g., the heavy chain) library. The second coding region codesfor an amino acid sequence that anchors and/or binds to cell membrane.The first and second coding regions are in-frame and operatively linkedso as one polypeptide is formed during translation. This results in afusion protein comprising the heavy chain of an antibody or antibodyfragment and an amino acid sequence that anchors and/or binds to thecell membrane. In one embodiment, the first and second coding regionsmay be directly linked/adjacent to one another. In another embodiment,at least one codon is between or separates the first and second codingregions. In one embodiment, the at least one codon provides a linker orspacer sequence between the antibody chain (e.g., heavy chain) and themembrane anchoring/binding domain. In one embodiment, the second codingregion codes for an amino acid sequence that anchors and/or binds tocell membrane, or codes for a transmembrane domain or a GPI-anchor.

With respect to the viral vector library, said first coding region ofthe library will be a genetically diverse repertoire of nucleic acidsequences which each encode a heavy chain of an antibody or antibodyfragment. In one embodiment, the second coding region is the samethroughout the library (e.g., a GPI-anchor domain from decayaccelerating factor (DAF).

In one embodiment, the first coding region is operatively linked to apromoter. In one embodiment, the promoter is heterologous in relation tothe viral vector. For example, the heterologous promoter is not derivedfrom the same virus that the viral vector is derived from. In anotherembodiment, the promoter is not heterologous. For example, the promoteris derived from the same virus as the viral vector is derived from.

The promoter can essentially be any promoter that is active or can beinduced to be active in the chosen cell. Although, the type of viralvector may influence the selection of promoters. For example, it isdesirable to avoid promoters that may interfere with the particularviral vector. For example, copies of the same promoter in an adenoviralvector may lead to homologous recombination during replication of thevector (e.g., see Stecher et al., 2003, Methods Mol Med. 76:135-52;Carlson et al., 2002, Methods Enzymol. 346:277-92) Conversely, it isdesirable to avoid promoters, where a particular viral vector mayinterfere with the promoter (e.g., see Grave et al., 2000, J Gene Med.2:433-43).

Promoters which may be used to control the expression of the firstcoding region encoding an antibody heavy chain or fragment include, butare not limited to, the SV40 early promoter region (Bemoist and Chambon,1981, Nature 290:304-310), a CMV promoter, the promoter contained in the3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al., 1980,Cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al.,1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatorysequences of the metallothionein gene (Brinster et al., 1982, Nature296:39-42), the tetracycline (Tet) promoter (Gossen et al., 1995, Proc.Nat. Acad. Sci. USA 89:5547-5551); the ADC (alcohol dehydrogenase)promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatasepromoter, the immunoglobulin gene control region which is active inlymphoid cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.7:1436-1444), mouse mammary tumor virus control region which is activein testicular, breast, lymphoid and mast cells (Leder et al., 1986, Cell45:485-495), albumin gene control region which is active in liver(Pinkert et al., 1987, Genes and Devel. 1:268-276), alpha-fetoproteingene control region which is active in liver (Krumlauf et al., 1985,Mol. Cell. Biol. 5:1639-1648; Hammer et al., 1987, Science 235:53-58;alpha 1-antitrypsin gene control region which is active in the liver(Kelsey et al., 1987, Genes and Devel. 1:161-171), beta-globin genecontrol region which is active in myeloid cells (Mogram et al., 1985,Nature 315:338-340; Kollias et al., 1986, Cell 46:89-94; myelin basicprotein gene control region which is active in oligodendrocyte cells inthe brain (Readhead et al., 1987, Cell 48:703-712); myosin light chain-2gene control region which is active in skeletal muscle (Sani, 1985,Nature 314:283-286); neuronal-specific enolase (NSE) which is active inneuronal cells (Morelli et al., 1999, Gen. Virol. 80:571-83);brain-derived neurotrophic factor (BDNF) gene control region which isactive in neuronal cells (Tabuchi et al., 1998, Biochem. Biophysic. Res.Com. 253:818-823); glial fibrillary acidic protein (GFAP) promoter whichis active in astrocytes (Gomes et al., 1999, Braz J Med Biol Res 32(5):619-631; Morelli et al., 1999, Gen. Virol. 80:571-83) and gonadotropicreleasing hormone gene control region which is active in thehypothalamus (Mason et al., 1986, Science 234:1372-1378).

In one embodiment, a polyadenylation signal is located 3′ to andoperatively linked to the second coding region. Essentially anypolyadenylation signal that is active in the particular cell typecontaining the nucleic acid may be utilized. Although, the type of viralvector may influence the selection of polyadenylation signal.Polyadenylation signals that may find use with the present inventioninclude, but are not limited to, those from SV40 and the bovine growthhormone gene.

In one embodiment, the nucleic acid also comprises a coding region foran amino terminus signal peptide sequence located upstream (5′) of andoperatively linked to the first coding region. Without wishing to belimited by theoretical considerations, the signal peptide directs theprotein for initial transfer into the endoplasmic reticulum (ER). In oneembodiment, the signal sequence at the amino terminus of the protein iscleaved during post-translational processing of the protein. In oneembodiment, a native signal sequence is retained. In another embodiment,the native signal sequence is deleted and replaced with a heterologoussignal sequence. The heterologous signal sequence selected should be onethat is recognized and optionally processed (i.e. cleaved by a signalpeptidase) by the particular host cell.

In one embodiment, the viral vector nucleic acid further comprises acoding sequence for an antibody light chain or fragment of an antibodylight chain. As shown in FIGS. 1C and 1E, the light chain codingsequence can be operatively linked to a promoter different from thepromoter operative linked to the heavy chain coding sequence. In otherwords, the expression of the light chain is not directly linked to theexpression of the heavy chain. In another embodiment, as shown inExamples IV and VI of FIG. 1, the light chain coding sequence isoperatively linked to the same promoter as the heavy chain codingsequence by either an internal ribosome entry site (IRES) or aself-processing cleavage sequence.

The vectors depicted in examples I-VI of FIG. 1A-C depict nonlimitingexamples of viral vector constructs of the invention.

7.3 Viral Vector Library Construction

Viral vector libraries of the present invention can be constructed byany number of methods know to those skilled in the art. Essentially anyantibody library can be cloned into a viral vector and be utilized inaccordance with the present invention. Briefly, a library of nucleicacids coding for a diverse repertoire of nucleic acid sequences whicheach encode a heavy chain of an antibody or antibody fragment areisolated. For example, the repertoire of nucleic acid sequences codingfor an antibody heavy chain can be isolated from, but not limited to, anantibody cDNA library, a cDNA library generated from nucleic acids,e.g., poly A+RNA, isolated from any tissue or cells expressingantibodies. The repertoire of coding sequences can then be amplified,for example by PCR, and cloned into viral vectors (e.g. replicable viralvectors) using standard methods known in the art. Libraries of antibodycoding sequences are also commercially available. In one embodiment, thelibrary is constructed using coding regions from human antibodies. Insome embodiments, the library expresses at least 100, 10³, 10⁴, 10⁵,10⁶, 5×10⁶, 10⁷, 5×10⁷, 10⁸, 5×10⁸, 10⁹, 5×10⁹, 10¹⁰, 5×10¹⁰, 10¹¹,5×10¹¹ or 10¹² different antibodies.

Once a library of nucleic acids coding for an antibody heavy chain isobtained, it is cloned into a viral vector. Essentially any methodsknown for cloning nucleic acids into a viral vector can be utilized.These methods include, but are not limited to, restriction enzymedigestion and ligation, pcr SOEing (Horton, et al., 1989, Gene, 77,61-68)or recombination. In one embodiment, the viral vector is anadenoviral vector and the cloning method is recombination. Recombinationcan be performed by any method known in the art. In one embodiment, theBJ5183 recombination method/system is utilized (for examples see PCTPatent Publication Nos. WO 02/067861 and WO 96/17070). Briefly, thelibrary of coding regions is cloned into a plasmid resulting in thecoding regions being flanked by sequences homologous to the region ofinsertion into the adenoviral vector. The plasmid library isco-transformed into BJ5183 cells with a compatible adenoviral vectorplasmid. Full-length adenoviral vector plasmids containing the insertionare isolated and transfected into mammalian cells to produce theadenoviral vector library.

Another recombination method for constructing an adenoviral vectorlibrary utilizes the Gateway® system from Invitrogen (Carlsbad, Calif.),for example using the ViraPower™ Adenoviral Gateway® Vectors. Thissystem uses a plasmid that contains the complete DNA sequence of anadenoviral vector. The adenoviral vector contains deletions in the E1and E3 coding regions. The adenoviral vector can be propagated in 293Acells (Invitrogen, CA) that express the E1 proteins. E3 is anonessential region for adenovirus replication in vitro.

Protocols for cloning nucleic acids into this system are available fromInvitrogen. Briefly and by way of example, a method for constructing anadenoviral vector library using this system is described as follows. Thelibrary of nucleic acids is cloned into an entry vector creating anentry clone. Entry vectors include, but are not limited to,pENTR™/D-TOPO®; pENTR™/SD/D-TOPO®; pENTR™/TEV/D-TOPO®; pENTR™1A;pENTR™2B; pENTR™3C; pENTR™4; and pENTR™11 (all available fromInvitrogen, CA). These vectors contain a multiple cloning site (MCS)flanked by recombination sites (e.g., attR1 and attR2). The library ofnucleic acids is cloned into the MCS, which results in the individuallibrary sequences being flanked by the recombination sites. The nextstep is to clone the library from the entry vector into an adenoviralvector plasmid (e.g., pAd/PL-DEST™ or pAd/CMV/V5-PL-DEST™ (bothavailable from Invitrogen)). This cloning step utilizes recombinationbetween the recombination sites in the entry vector and those in theadenoviral vector plasmid to create an adenoviral vector plasmidlibrary. To produce the viral vector particles the recombined adenoviralvector plasmid library is digested with PacI and transfected into the293A cells. The virus is then amplified and optionally purified,creating a stock of an adenoviral vector library. For further detailssee the following Invitrogen™ instruction manuals: pAd/CMV/V5-PL-DEST™and pAd/PL-DEST™ Gateway® Vectors, Version D, Sept. 28, 2005; ViraPower™Adenoviral Expression System, Version B, Jul. 11, 2005.

The methods of screening cells displaying antibodies as describedherein, can be utilized in combination with other antibody screeningmethods and/or systems. For example, one embodiment of the inventionutilizes mammalian cells expressing a library of antibodies on the cellsurface. This library can be from any source. It can be a large libraryisolated from human cells and essentially represent a complete humanrepertoire of antibodies variable regions from a subject or subjects. Inanother embodiment, the library may be isolated from a mouse (e.g. amouse expressing human antibodies) that has been immunized with theantigen of interest. Therefore, the library is enriched for antibodiesthat bind the antigen of interest. In another embodiment, a phagedisplay antibody library is screened against the antigen of interest.The antibody library for the present invention is then created fromthose phage that express an antibody that binds the antigen of interest.Again, the library is enriched for antibodies that bind the antigen ofinterest. In still another embodiment, an antibody library for thepresent invention is generated from a library of humanized antibodyfragments. Humanized antibody fragments may be generated by any methodknown to one of skill in the art including, but not limited to,framework shuffling (e.g., PCT Publication WO 05/042743) and lowhomology humanization (e.g., PCT Publication WO 05/035575). In anotherembodiment, the library is a mutant CDR library derived from an antibodythat binds the antigen of interest. A mutant CDR library is a librarycoding for antibodies that are CDR mutations of a parent antibody's CDRsequences. Mutant CDR include, but are not limited to, libraries createdby mutating CDR amino acids that are determined to be contact residuesby crystallographic studies (e.g., Dall'Acqua et al. 1996 Biochemistry35:9667-76); libraries created by retaining one native CDR (e.g. the onebelieved to have the highest binding efficiency) and combining with alibrary of CDRs in place of the other 5 CDRs (e.g., Rader et al., 1998,PNAS 95:8910-15); a library created by “CDR walking” (e.g., Yang et al.,1995, J Mol Biol 254:392-403; and a library created by a method ofseparately mutating each CDR of a parental antibody (e.g., Wu et al.,1998, PNAS 95:6037-42). Therefore, any library of antibodies may be usedin accordance with the present invention to express the library on thecell membrane. In one embodiment, the library is an affinity maturationlibrary derived from a parental antibody.

In one embodiment, a phage display antibody library is screened againstthe antigen of interest. The antibody library for the present inventionis then created from those phage that express an antibody that binds theantigen of interest. In one embodiment, both the heavy chain and lightchain variable regions from each selected phage are cloned into a viralvector (e.g. adenoviral vector), wherein each viral vector encodes aheavy and light chain. Therefore, the same heavy and light chaincombinations are maintained. In another embodiment, the heavy chain andlight chain variable regions are isolated and combined in a randommatter. Therefore, theoretically the library comprises every combinationof each preselected heavy chain variable sequence with each preselectedlight chain sequence. For example, if the initial phage screen resultedin 1,000 unique phage and antibody sequences, a library comprised ofevery combination of each preselected heavy chain variable sequence witheach preselected light chain sequence would now account for 10⁶ possibleunique antibody sequences that would be cloned into a viral vector ofthe invention. This method creates an even more diverse repertoire thanthe initial phage library. Additionally, the phage display method islimited to certain antibody fragments, whereas this method allows aninitially selected screen in phage of antibody fragments and asubsequent screen of, for example, whole antibodies created from thevariable region sequences isolated from the phage selection step.Examples of phage display methods that can be used to make the antibodylibraries of the present invention include those disclosed in Brinkmanet al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J.Immunol. Methods 184:177-186; Kettleborough et al., 1994, Eur. J.Immunol. 24:952-958; Persic et al., 1997, Gene 187:9-18; Burton et al.,1994, Advances in Immunology 57:191-280; PCT Publication Nos. WO90/02809, WO 91/10737, WO 92/01047, WO 92/18619, WO 93/11236, WO95/15982, WO 95/20401, and WO97/13844; and U.S. Pat. Nos. 5,698,426,5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047,5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743 and5,969,108.

For further details and methods for cloning antibody libraries see e.g.,PCT Patent Publication WO 2005/063817; WO 95/15393; and Higuchi et al.1997 J Immunological Methods 202:193-204.

7.4 Selection Strategies & Methods

Once a viral vector antibody library is constructed, it can be screenedagainst at least one antigen of interest. It will be appreciated bythose skilled in the art that numerous variations for screening may bemade without departing from the invention as described herein.

Generally, cells are infected with the viral vector antibody library. Anappropriate MOI will be used based on several factors including, but notlimited to, the number of cells available; the number of cells that canor are desired to be screened; titer of the viral vector, theinfectivity of the cells by the viral vector; and the toxicity of viralvector infection on the cells. Generally, the infection procedure,including the MOI, will be optimized prior to the screening of thelibrary.

After infection cells are cultured to allow expression of the antibodylibrary, which is displayed on the cell surface. The cells are thenscreened for those expressing an antibody that binds the antigen ofinterest or has desired characteristics (e.g., binding to Fc receptors).The cells can be screened by methods described herein and those known toone skilled in the art. Cells expressing antibodies with the desiredproperties are separated from the other cells.

At this point the nucleic acids encoding the positive antibodies may beisolated and used to express the corresponding antibodies for furthercharacterization. Alternatively, the viral vectors expressing thedesired antibodies can be isolated and put through another round ofscreening. In this second round of screening, the same or a differentMOI may be utilized. In one embodiment, the second and subsequent roundsof screening utilize MOIs lower that the initial infection. In oneembodiment, in each subsequent round of screening the MOI is decreased.Although applicants do no wish to be bound by mechanistic speculation,only a small percentage of unique individual members of the initiallibrary will be selected in the initial screening method. The higher theinitial MOI of the initial infection, may lead to a larger number ofirrelevant antibodies being selected. This is because the initialinfection with a high MOI will likely result in multiple viral vectorsinfecting and expressing an antibody in each cell. Therefore, theselected cell would only have to express one antibody that binds theantigen to be selected, but any other viral vectors expressingirrelevant antibodies in the same cell would also be selected. Thereforein most cases, it is desirable to perform at least a second round ofselection using a lower MOI. In some embodiments of the invention, asecond selection step utilizes an MOI of at most: 10⁻⁶, 10⁻⁵, 10⁻⁴,10⁻³, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 or 10.Lower MOIs can be used to virtually guarantee that each cell is infectedby 1 or less viral vectors. This eliminates “carry-over” sequences orviral vectors.

Once viral vectors encoding antibodies with the desired characteristicsare identified, the coding regions for these antibodies can be clonedinto other expression vectors/systems for further evaluation and/or theproduction of the antibodies. Selected antibodies can also be modifiedby methods known in the art and those described herein.

Once a library of cells expressing antibodies on the cell surface isconstructed, the next step is to screen and select for the cellsexpressing an antibody that binds a desired antigen or to select forantibodies with a desired characteristic (e.g., altered binding affinityfor an Fc receptor). This screening and selection step can beaccomplished using any of a variety of techniques known in the artincluding those described herein. For example, the antigen of interestmay be tagged (e.g. fluorescent marker) and used to bind to antibodieson the cell surface; thus labeling the cells expressing antibodies thatbind to the antigen. Numerous fluorescent labels are known in the artand commercially available (see, e.g., Molecular Probes: Handbook ofFluorescent Probes and Research Chemicals, R. P. Haugland, 9th ed.,Molecular Probes, (OR, 2004)).

In one embodiment, the antigen is biotin-labeled. The cells bind theantigen and a label conjugated to streptavidin is used to label cellsbound to the antigen. In one embodiment, PE-conjugated streptavidin isused. In one embodiment, the antigen comprises streptavidin and labeledbiotin is used as the detection reagent. These labeled cells can beidentified and isolated using techniques known in the art. For examplein the case of a fluorescently tagged antigen, the cells can beseparated/sorted, for example, by a flow cytometer and sorted based onfluorescence. For examples, see PCT Publication Nos. WO 04/014292, WO03/094859, WO 04/069264, WO 04/028551, WO 03/004057, WO 03/040304, WO00/78815, WO 02/070007 and WO 03/075957, U.S. Pat. Nos. 5,795,734,6,248,326 and 6,472,403, Pecheur et al., 2002, FASEB J. 16: 1266-1268;Almed et al., 2002, J. Histochemistry & Cytochemistry 50:1371-1379. Inanother embodiment, fluorescent cells are observed using a fluorescentmicroscope and may be isolated directly using standard micromanipulationtechniques such as a fine glass pipette, micropipettor or amicromanipulator. In another embodiment, the cells can besorted/separated using beads (e.g., magnetic beads; see Chestnut et al.,1996, J Immunological Methods 193:17-27). For example, an antigen can bebiotinylated, and cells expressing antibodies that bind to the antigencan be isolated using streptavidin-coated beads.

In one embodiment, the antigen is fluorescently labeled. In oneembodiment the fluorescent label is selected from the group consistingof Aqua, Texas-Red, FITC, rhodamine, rhodamine derivatives, fluorescein,fluorescein derivatives, cascade blue, Cy5, phycoertythrin, GFP or a GFPderivative e.g., EGFP.

In one embodiment, the antigen is recombinantly produced andincorporates a peptide tag, e.g., FLAG, HIS tag, Antibodies to thepeptide tag can be used to detect and sort/select for or exclude cellsthat bind the particular antigen.

More than one antigen may be utilized in the selection step, forexample, if screening for antibodies that bind a first antigen but not asecond antigen. In this case, a negative selection step could be carriedout by sorting for cells expressing antibodies that do not bind thesecond antigen, followed by a positive selection step that sorts forantibodies that bind the first antigen. In one embodiment, the positiveselection step is carried out prior to the negative selection step. Inone embodiment, the positive and negative selection step is carried outessentially simultaneously. For example, first and second antigen islabeled with different fluorescent molecules. Both antigens areincubated together with the cells displaying the antibodies. Theconcentration of the antibodies may be optimized for this embodiment.The cells are then simultaneously sorted for those that bind the first,but not the second antigen (e.g., two-color FACS analysis). One skilledin the art, based on the teachings herein, can negatively and/orpositively screen for binding to a multitude of antigens by employingconsecutive screening/selection steps and by multi-color FACS analysis.

In some embodiments antibodies that bind to a particular cell type(target cell) can be selected. Such selections in relation tophage-displayed antibodies are described in e.g. Huts et al., 2001,Cancer Immunol. Immunother. 50:163-171. The target cells can be fixed orunfixed, which may for example, offer an opportunity to selectantibodies that bind to cell surface antigens that are altered byfixation. A particular cell type can be selected with reference to thebiological function to be screened/selected for in the process. Anappropriate cell type would be one that antibodies with the desiredbiological function would be expected to bind. For example, if it isdesired to isolate antibodies capable of inhibiting the proliferation ofcancer cells, it would be expected that such antibodies could bind tocancer cells. Thus, it would be appropriate to initially select forantibodies that can bind to cancer cells. To select for antibodies thatbind to cells, the cells displaying antibodies can be screened forbinding to the target cell using conditions conducive to binding. Forexample, a biotin-conjugated antibody that binds to the target cells,but not to the cells expressing the antibodies, can be bound tostreptavidin-coated magnetic beads. These beads are then used toimmobilize the target cells. The antibody-expressing cells can becombined with the immobilized cells, and those that bind to the magneticbeads can be isolated. Selection for antibodies that bind to cells,rather than specific, known antigens, has the advantage that there is apossibility of selecting for antibodies that bind to previously unknownantigens displayed on a cell surface. Such an antigen need not be aprotein and may comprise more than one cell surface molecule. Aselection step for binding to a chosen kind of cells or a particularmolecule can be repeated once or multiple times, for example, at leastabout 2, 3, 4, 5, 6, or 7 times. If desired, two or more differentpre-selection steps can be performed either simultaneously or insuccession. For example, antibodies that bind to two different kinds ofcancer cells can be selected.

Optionally, further refinement can be achieved by one or more negativeselection steps, which can be performed either before or after thepositive selection step. For example, if selecting for antibodies thatbind to cancer cells, the cells displaying antibodies can be allowed tobind non-cancerous cells (e.g. as described above), and antibodies thatdo not bind to these cells can be retained for further testing. Such anegative selection can eliminate at least some of the antibodies thatbind nonspecifically to non-target cells. Alternatively, the non-targetprotein(s) (e.g. unrelated or similar antigen as compared to the targetantigen) is affixed to a solid support and utilized in a negativeselection step to eliminate antibody expressing cells that bind to thenon-target protein(s). In another example, it may be desired to isolateantibodies to a certain receptor, wherein this receptor is part of afamily of receptors that have closely related structures. To increasethe probability of isolating an antibody specific to this particularreceptor, a negative selection step may be performed using one, some orall of the other receptors from the family. In a negative selectionstep, cells expressing antibodies that do not bind the non-targetantigen can be retained for further testing. This selection caneliminate at least some of the antibodies that bind nonspecifically tothe solid support or to a non-target protein(s). Similarly, if selectingfor cells displaying antibodies that bind to a particular protein, thecells can be mixed with an unrelated or similar protein to compete forbinding with the target protein.

Screening methods of the present invention may employ 1, 2, 3, 4, 5, 6,7, 8 or more selection steps. The method used to perform secondaryscreens may depend on the viral vector being employed. Using anadenoviral vector for example, the viral vector can be directly isolatedfor subsequent rounds of screening/selection. For example, the cells(e.g. 293 based cells) are initially infected with the viral vectorlibrary followed by selection of cells that express antibodies that bindthe antigen of interest. The cells are then lysed to release thepackaged viral vectors. The cells can be lysed by any number of methods(e.g. freeze thawing the cells once or multiple times) that do noteliminate the infectivity of the viral vector. The cell lysate can beused directly for the infection in the next round of screening/selectionor the viral vectors can be purified first.

In the case of adenoviral vectors, the MOI of infection, time ofscreening/sorting and time of isolating the viral vectors may need to beconsidered. For example, if the MOI is too high, the cells may lysebefore the screening/sorting method. Additionally, if thescreening/sorting step is performed to soon after the infection, theantibody may not yet be displayed on the cell surface or in sufficientquantity. If the screening step is performed too late, the cells maylyse from viral vector toxicity (e.g. replication). If the isolation ofthe vector is attempted too early, there may not be enough viral vectorpackaged. Adenoviral vectors have been used for decades for geneexpression and optimizing these parameters are well within the skill ofthose in the art.

One skilled in the art can readily identify cells that could be used inaccordance with the present invention. Cells that can be used forexpressing the cell displayed antibodies include, but are not limitedto, CHO, BHK, HeLa, COS, COS7, MDCK, TM4, CV1, VERO, BRL 3A, Hep G2; MMT060562; TRI; MRC5; FS4; NIH 3T3, W138, NSO, SP/20 and other lymphocyticcells, and human cells such as PERC6, HEK 293, 293A, breast cancer celllines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, andnormal mammary gland cell line such as, for example, CRL7030 andHsS78Bst. In one embodiment the cells are a 293 derivative. In oneembodiment, the cells express at least one E1 adenoviral protein and theE1 coding region is integrated into the cellular genome.

When cells and/or viral vectors expressing antibodies with the desiredproperties are identified in the preceding steps, nucleic acids encodingthem can be isolated and retested to ensure that they encode antibodieswith the desired biological properties. If individual transformants orpools of transformants are isolated, recombinant nucleic acids can beobtained from these for retesting. For example, if individualtransformants have been isolated, nucleic acids encoding the antibodiescan be purified and used to transfect mammalian cells, which can then becharacterized with regards to their binding properties for the antigen.If pools of transformants have been isolated, nucleic acids encoding theantibodies from pools testing positive can be used to transform cells togenerate individual transformants expressing one antibody.

Nucleic acids encoding the antibodies from these individualtransfonnants can be used to transfect cells and antibodies can beexpressed, isolated and tested for function, thereby identifyingproteins or antibodies having the desired function. If individualtransformants or pools of transformants have not been isolated, nucleicacids encoding the protein or at least the antibody variable regions canbe obtained from the transfectants or pools of transfectants that havetested positive, for example, by amplifying the expressed antibodyvariable region-encoding sequences by PCR. These sequences, which may beamplified by PCR, can also then be re-inserted into a suitable vectorand used to generate individual transformants. Recombinant DNA fromthese transformants can be used to transfect mammalian cells in orderexpress the antibodies and to retest for function.

7.5 Screening Based on Characteristics of Fc Receptor/Ligand Bindingand/or Antibody Dependent Cell-Mediated Cytotoxicity (ADCC) and/orComplement Dependent Cytotoxicity (CDC)

The Fc region of an antibody interacts with a number of ligandsincluding Fc receptors and other ligands, imparting an array ofimportant functional capabilities referred to as effector functions. Animportant family of Fc receptors for the IgG class is the Fc gammareceptors (FcγRs). These receptors mediate communication betweenantibodies and the cellular arm of the immune system (Raghavan et al.,1996, Annu Rev Cell Dev Biol 12:181-220; Ravetch et al., 2001, Annu RevImmunol 19:275-290). In humans this protein family includes FcγRI(CID64), including isoforms FcγRIA, FcγRIB, and FcγRIC; FcγRII (CD32),including isoforms FcγRIIA, FcγRIIB, and FcγRIIC; and FcγRIII (CID16),including isoforms FcγRIIIA and FcγRIIB (Jefferis et al., 2002, ImmunolLett 82:57-65). These receptors typically have an extracellular domainthat mediates binding to Fc, a membrane spanning region, and anintracellular domain that may mediate signaling events within the cell.Different FcγR subtypes are expressed on different cell types (reviewedin Ravetch et al., 1991, Annu Rev Immunol 9:457-492). For example, inhumans, FcγRIIIB is found only on neutrophils, whereas FcγRIIIA is foundon macrophages, monocytes, natural killer (NK) cells, and asubpopulation of T-cells.

Formation of the Fc/FcγR complex recruits effector cells to sites ofbound antigen, typically resulting in signaling events within the cellsand important subsequent immune responses such as release ofinflammation mediators, B cell activation, endocytosis, phagocytosis,and cytotoxic attack. The ability to mediate cytotoxic and phagocyticeffector functions is a potential mechanism by which antibodies destroytargeted cells. The cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell is referred to as antibodydependent cell-mediated cytotoxicity (ADCC) (Raghavan et al., 1996, AnnuRev Cell Dev Biol 12:181-220; Ghetie et al., 2000, Annu Rev Immunol18:739-766; Ravetch et al., 2001, Annu Rev Immunol 19:275-290). Notably,the primary cells for mediating ADCC, NK cells, express only FcγRIIIAonly, whereas monocytes express FcγRI, FcγRII and FcγRIII (Ravetch etal., 1991, ibid).

Another important Fc ligand is the complement protein C1q. Fc binding toC1q mediates a process called complement dependent cytotoxicity (CDC)(reviewed in Ward et al., 1995, Ther Immunol 2:77-94). C1q is capable ofbinding six antibodies, although binding to two IgGs is sufficient toactivate the complement cascade. C1q forms a complex with the C1r andC1s serine proteases to form the C1 complex of the complement pathway.

All FcγRs bind the same region on the Fc of the IgG subclass, but withdifferent affinities (e.g., FcγRI is a high affinity while FcγRII andFcγRIII are low affinity binders). Other differences between the FcγRsare mechanistic. For example, FcγRI, FcγRIIA/C, and FcγRIIIA arepositive regulators of immune complex triggered activation,characterized by having an immunoreceptor tyrosine-based activationmotif(ITAM) while FcγRIIB has an immunoreceptor tyrosine-basedinhibition motif(ITIM) and is therefore inhibitory. Thus, the balancebetween activating and inhibiting receptors is an importantconsideration. For example, enhancing Fc binding to the positiveregulators (e.g., FcγRIIIA) while leaving unchanged or even reducing Fcbinding to the negative regulator FcγRIIB could result in optimizedeffector function such as enhanced ADCC mediated destruction of tumorcells. Another consideration is that Fc variants should be engineeredsuch that the binding to FcγRs and/or C1q is modulated in the desiredmanner but so that they maintain their stability, solubility, structuralintegrity as well as their ability to interact with other important Fcligands such as FcRn and proteins A and G.

Antibodies find utility in a number of applications includingtherapeutic uses. Depending on the application, the desired ADCC and/orCDC characteristics of the antibody may vary. For example, in diagnosticapplication (e.g. ELISA, Western Blot, etc.,) the ADCC and/or CDCactivity of the antibody is usually irrelevant and has little effectupon the diagnostic application. In the case of using anti-tumor antigenantibodies, increased ADCC and/or CDC may be desired to increase the invivo or even in vitro cytotoxicity and therefore increase the potency ofthe antibody in relation to killing the tumor cells. In applications,for example, where the antibody is used in vivo to as an antagonist oragonist, it may be desired to use antibodies with decreased, low or noCDC and/or ADCC activity. This is particularly true for those antibodiesdesigned to deliver a drug (e.g., toxins and isotopes) to the targetcell where the Fc/FcγR mediated effector functions bring healthy immunecells into the proximity of the deadly payload, resulting in depletionof normal lymphoid tissue along with the target cells (Hutchins et al.,1995, PNAS USA 92:11980-11984; White et al., 2001, Annu Rev Med52:125-145). In these cases the use of Fc variants that poorly recruitcomplement or effector cells would be of tremendous benefit (see forexample, Wu et al., 2000, Cell Immunol 200:16-26; Shields et al., 2001,J. Biol Chem 276:6591-6604; U.S. Pat. No. 6,194,551; U.S. Pat. No.5,885,573 and PCT Patent Publication WO 04/029207). Accordingly, thepresent invention additionally provides methods of screening antibodylibraries based on Fc receptor (e.g., FcγR) and/or Fc ligand (e.g., C1q)binding and/or effector function (e.g., ADCC activity). The presentinvention can be utilized to screen for antibodies and/or antibodyvariants with any of these characteristics or combinations thereof.

Various mutagenesis studies have been carried out on the Fc domain (Seefor example, Duncan et al., 1988, Nature 332:563-564; Lund et al., 1995,Faseb J 9:115-119; Lund et al., 1996, J Immunol 157:4963-4969; Armour etal., 1999, Eur J Immunol 29:2613-2624; Shields et al., 2001, J Biol Chem276:6591-6604; Jefferis et al., 2002, Immunol Lett 82:57-65; Presta etal., 2002, Biochem Soc Trans 30:487-490; U.S. Pat. Nos. 5,624,821,5,885,573 and PCT Patent Publication Nos. WO 00/42072, WO 99/58572 andWO 04/029207). While the vast majority of amino acid substitutions inthe Fc domain reduce or ablate FcγR binding, some have resulted inhigher affinity for FcγR. For examples of specificmodifications/substitutions and/or novel amino acids within the Fcdomains see: Ghetie et al., 1997, Nat Biotech. 15:637-40; Duncan et al,1988, Nature 332:563-564; Lund et al., 1991, J. Immunol 147:2657-2662;Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al, 1994,Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad SciUSA 92:11980-11984; Jefferis et al, 1995, Immunol Lett. 44:111-117; Lundet al., 1995, Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett54:101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al.,1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol164:4178-4184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu et al.,2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferiset al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem SocTrans 30:487-490); U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425;6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260;6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. patent application Ser.Nos. 10/370,749; 11/203,253; 11/203,251 and PCT Publications WO 94/2935;WO 99/58572; WO 00/42072; WO 01/58957; WO 02/060919, WO 04/016750; WO04/029207; WO 04/035752 and WO 05/040217.

Fc regions comprising at least one amino acid substitution (i.e., a nonnaturally occurring amino acid residue), deletion or insertionintroduced at any position within the Fc region are referred to hereinas “variant Fc regions”. Polypeptides comprising variant Fc regions(e.g., antibodies or Fc fusion proteins) are referred to hereingenerally as “Fc variants” or more specifically as “Fc variantantibodies” and “Fc variant fusion proteins.” It is contemplated thatthe cell surface display methods disclosed herein may be utilized forexpressing and screening a library of antibodies with variant Fcregions.

Libraries comprising antibodies with variant Fc regions (also referredto herein as “Fc variant libraries”) may be screened for the desired Fcrelated function or lack thereof in accordance with the presentinvention. In one embodiment, the library of Fc variants comprisesantibodies containing the same variable regions or Fab regions. In someembodiments, the library contains variants of the hinge domain, CH3domain, CH2 domain or any combination thereof.

Methods for constructing Fc variants and Fc variant antibody librariesare know in the art. For examples, see Patent Publication Nos. WO05/0037000; WO 06/023420; and WO 06/023403. It is contemplated that anFc variant library comprises Fc regions with at least one amino acidsubstitution, deletion or insertion introduced at any position withinthe Fc region. It is also contemplated that an Fc variant library mayfurther comprise additional amino acid residue substitutions (i.e., anon naturally occurring amino acid residue), deletions or insertions atone or more positions outside of the Fc region. In certain embodiments,an Fc variant library comprises Fc regions with at least onenon-naturally occurring amino acid residue at any position within the Fcregion. In specific embodiments, an Fc variant library comprises Fcregions that comprise at one or more position within the Fc region eachof the 19 non-naturally occurring amino acid residues. In other specificembodiments, the Fc variant library comprises Fc regions that compriseat one or more position within the Fc region a subset of non-naturallyoccurring amino acid residues. In still other embodiment, the Fc variantlibrary comprises Fc region that comprise the insertion of one or moreamino acid residue at one or more position.

Without wishing to be bound by any particular theory, the amino acidsubstitutions (i.e., a non naturally occurring amino acid residue),deletions and/or insertions of the invention modulate the ADCC and/orCDC activity of an antibody by altering one or more of the factors thatinfluence downstream effector function, including but not limited to,the affinity of the antibody for its FcγRs and/or to C1q, ability tomediate cytotoxic effector and/or complement cascade functions, proteinstability, antibody half life and recruitment of effector cells and/ormolecules.

In one embodiment, the library comprises Fc variants with at least oneamino acid residue substitution (i.e., a non naturally occurring aminoacid residue), deletion or insertion at a position selected from thegroup consisting of amino acid residues: 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 242, 246, 250, 251, 256, 257, 259, 260, 261,265, 266, 269, 273, 274, 275, 277, 281, 282, 298, 327, 328, 329, 330,332, 346, 347 and 348, wherein the numbering system is that of the EUindex as set forth in Kabat et al. (1991, NIH Publication 91-3242,National Technical Information Service, Springfield, Va.).

In another embodiment of the invention, the Fc variant librarycomprises, Fc variants having each of the non naturally occurring aminoacid residues, at one or more of the amino acid residues at: 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 242, 246, 250, 251, 256,257, 259, 260, 261, 265, 266, 269, 273, 274, 275, 277, 281, 282, 298,327, 328, 329, 330, 332, 346 and 348, wherein the numbering system isthat of the EU index as set forth in Kabat.

In one embodiment, the Fc variants of the library comprise at least oneamino acid substitution (i.e., a non naturally occurring amino acidresidue), deletion or insertion at a position selected from the groupconsisting of: 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216,217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230,231, 232, 233, 234, 235, 236, 237, 239, 240, 242, 246, 250, 251, 257,259, 260, 261, 265, 269, 273, 274, 275, 277, 281, 282, 284, 287, 291,298, 300, 302, 304, 306, 308, 310, 314, 316, 318, 319, 321, 323, 327,328, 329, 330, 332 and 336, wherein the numbering of the residues in theFc region is that of the EU index as set forth in Kabat. In oneembodiment, the library comprises Fc variants comprising at least 2, orat least 3, or at least 4, or at least 5, or at least 6, or at least 7,or at least 8, or at least 9, or at least 10, or at least 20, or atleast 30, or at least 40, or at least 50, or at least 60, or at least70, or at least 80, or at least 90, or at least 100, or at least 200amino acid residues in the Fc region.

In one embodiment, the Fc variants of the library comprise at least oneinsertion after a position selected from the group consisting of aminoacid residues: 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,242, 246, 250, 251, 256, 257, 259, 260, 261, 265, 266, 269, 273, 274,275, 277, 281, 282, 298, 327, 328, 329, 330, 332, 346 and 348, whereinthe numbering system is that of the EU index as set forth in Kabat. Theinsertion may be any amino acid residue. In certain embodiments an Fcvariant of the invention may comprises the insertion of more than oneamino acid residue after a selected position. In certain otherembodiments, an Fc variant of the invention may comprises the insertionof one or more amino acid residues after multiple positions.

In a specific embodiment, the Fc variants of the library comprise atleast one insertion after a position selected from the group consistingof amino acid residues: 230, 231, 232, 233, 234, 235, 236, 237, 238, 239and 240, wherein the numbering system is that of the EU index as setforth in Kabat. The insertion may be any amino acid residue. In certainembodiments an Fc variant of the invention may comprises the insertionof more than one amino acid residue after a selected position. Incertain other embodiments, an Fc variant of the invention may comprisesthe insertion of one or more amino acid residues after multiplepositions.

In other embodiments, the Fc variants of the library comprise acombination of a substitution and an insertion. In other embodiments,the Fc variants of the library comprise a combination of one or more ofthe substitutions and one or more of the insertions.

The present invention also provides Fc variants comprising at least oneamino acid residue substitution (i.e., a non naturally occurring aminoacid residue), deletion or insertion at a position selected from thegroup consisting of amino acid residues: 230, 231, 232, 233, 234, 235,236, 237, 238, 239, 240, 242, 246, 250, 251, 256, 257, 259, 260, 261,265, 266, 269, 273, 274, 275, 277, 281, 282, 298, 327, 328, 329, 330,332, 346 and 348, wherein the numbering system is that of the EU indexas set forth in Kabat.

In certain embodiments, an Fc variant of the present invention comprisesat least one non naturally occurring amino acid residue selected fromthe group consisting of: 231L, 231I, 231V, 231N, 231Q, 231T, 231S, 232K,232R, 234K, 234R, 235V, 235I, 235A, 235G, 236K, 236R, 236L, 236I, 236V,236A, 237R, 237K, 238N, 238Q, 238V, 238L, 238I, 238E, 238D, 238A, 238G,238M, 238C, 239D, 240G, 240A, 240H, 240D, 240E, 246R, 246E, 246D, 246W,246F, 246M, 246C, 250S, 250V, 250I, 250L, 251 A, 251G, 251E, 251D, 251V,251I, 256R, 256K, 260R, 260K, 260E, 260D, 261S, 261T, 266A, 266G, 274R,277V, 277I, 277L, 277S, 277T, 281S, 281T, 282F, 282W, 346R, 346K, 348Gand 348A, wherein the numbering system is that of the EU index as setforth in Kabat.

In a specific embodiment, an Fc variant of the present inventioncomprises at least one non naturally occurring amino acid residuesselected from the group consisting of: 231L, 231N, 231T, 232K, 234R,235V, 235A, 235I, 236R, 236V, 236A, 237R, 237G 238N, 238V, 238E, 238L,238G, 238M, 238Q, 239D, 240G, 240H, 240E, 246R, 246E, 246W, 246M, 250S,250V, 251A, 251E, 251I, 256R, 260R, 260E, 261S, 265, 266A, 274R, 277V,277T, 281S, 282F, 346R and 348A, wherein the numbering system is that ofthe EU index as set forth in Kabat.

In another specific embodiment, an Fc variant of the present inventioncomprises at least one non naturally occurring amino acid residuesselected from the group consisting of: 198T, 234R, 236R, 236A, 237R,238L, 238E, 238N, 238V, 238Q, 240E, 240G, 248E, 251A, 251E, 266A, 277T.In yet another specific embodiment, an Fc variant of the presentinvention comprises at least one combination of non naturally occurringamino acid residues selected from the group consisting of:246R/251E/260R, 240G/198T, 237R/236A.

In certain embodiments, an Fc variant of the present invention comprisesat least one insertion after a position selected from the groupconsisting of amino acid residues: 230, 231, 232, 233, 234, 235, 236,237, 238, 239, 240, 242, 246, 250, 251, 256, 257, 259, 260, 261, 265,266, 269, 273, 274, 275, 277, 281, 282, 298, 327, 328, 329, 330, 332,346 and 348, wherein the numbering system is that of the EU index as setforth in Kabat. The insertion may be any amino acid residue. Specificinsertions may be identified herein as “In” followed by the one lettercode of the inserted amino acid residue and the position of the residuesimmediately flanking the insertion. For example “InG231/232” denotes avariant Fc comprising an insertion of a glycine between residues 231 and232. In certain embodiments an Fc variant of the invention may comprisesthe insertion of more than one amino acid residue after a selectedposition. In certain other embodiments, an Fc variant of the inventionmay comprises the insertion of one or more amino acid residues aftermultiple positions.

In a specific embodiment, an Fc variant of the present inventioncomprises an insertion after a position selected from the groupconsisting of amino acid residues: 230, 231, 232, 233, 234, 235, 236,237, 238, 239 and 240, wherein the numbering system is that of the EUindex as set forth in Kabat. The insertion may be any amino acidresidue. In certain embodiments an Fc variant of the invention maycomprises the insertion of more than one amino acid residue after aselected position. In certain other embodiments, an Fc variant of theinvention may comprises the insertion of one or more amino acid residuesafter multiple positions.

In another specific embodiment, an Fc variant of the present inventioncomprises at least one of the following insertions: InR234/235;InV235/236; InR236/237; InR237/238; InV238/239; InN238/239; InL238/239;InE238/239; InG238/239; InS239/240; InG240/241 and InE240/241.

In certain embodiments, an Fc variant of the present invention maycomprise a combination of a substitution and an insertion. In otherembodiments, an Fc variant of the present invention may comprise acombination of one or more of the substitutions and one or more of theinsertions disclosed herein. In a specific embodiment, an Fc variant ofthe present invention comprises at least one of the followingcombinations of insertions and substitutions: InG240/241/I198T,InL238/239/P238Q, InE238/239/V348A, InS239/240/V266A, andInR237/238/G236A.

In one embodiment, the Fc variants of the present invention may becombined with other known Fc variants such as those disclosed in U.S.Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375;5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,194,551; 6,737,056;6,821,505; 6,277,375; U.S. patent application Ser. Nos. 10/370,749;11/203,253; 11/203,251 and PCT Publications WO 94/2935; WO 99/58572; WO00/42072; WO 01/58957; WO 02/060919, WO 04/016750; WO 04/029207; WO04/035752 and WO 05/040217.

It will be apparent to one skilled in the art that in addition to thespecific amino acid residues described above, a number of additionalamino acid residues may be inserted, deleted and/or substituted in thehinge to change the characteristics of the hinge. Families of amino acidresidues having similar properties have been defined in the art andseveral examples are shown in Table 1.

TABLE 1 Properties of Amino Acid Residues. Family Amino Acids non-polar(hydrophobic) Trp, Phe, Met, Leu, Ile, Val, Ala, Pro, Gly, unchargedpolar (hydrophilic) Ser, Thr, Asn, Gln, Tyr, Cys acidic/negativelycharged Asp, Glu basic/positively charged Arg, Lys, His Beta-branchedThr, Val, Ile residues that influence chain orientation Gly, Proaromatic Trp, Tyr, Phe, His

It is specifically contemplated that conservative amino acidsubstitutions may be made for said modifications of the hinge, describedsupra. It is well known in the art that “conservative amino acidsubstitution” refers to amino acid substitutions that substitutefunctionally equivalent amino acids. Conservative amino acid changesresult in silent changes in the amino acid sequence of the resultingpeptide. For example, one or more amino acids of a similar polarity actas functional equivalents and result in a silent alteration within theamino acid sequence of the peptide. Substitutions that are chargeneutral and which replace a residue with a smaller residue may also beconsidered “conservative substitutions” even if the residues are indifferent groups (e.g., replacement of phenylalanine with the smallerisoleucine). Families of amino acid residues having similar side chainshave been defined in the art. Several families of conservative aminoacid substitutions are shown in Table 1. (supra).

The term “conservative amino acid substitution” also refers to the useof amino acid analogs or variants. Guidance concerning how to makephenotypically silent amino acid substitutions is provided in Bowie etal., “Deciphering the Message in Protein Sequences: Tolerance to AminoAcid Substitutions,” (1990, Science 247:1306-1310).

The invention further encompasses incorporation of unnatural amino acidsin the modification of the hinge to generate the Fc variants of theinvention. Such methods are known to those skilled in the art such asthose using the natural biosynthetic machinery to allow incorporation ofunnatural amino acids into proteins, see, e.g., Wang et al., 2002 Chem.Comm. 1: 1-11; Wang et al., 2001, Science, 292: 498-500; van Hest etal., 2001. Chem. Comm. 19: 1897-1904. Alternative strategies focus onthe enzymes responsible for the biosynthesis of amino acyl-tRNA, see,e.g., Tang et al., 2001, J. Am. Chem. 123(44): 11089-11090; Kiick etal., 2001, FEBS Lett. 505(3): 465.

One skilled in the art will understand that that the Fc variant librarymay be screened for those Fc variants having altered FcγR and/or C1qbinding properties (examples of binding properties include but are notlimited to, binding specificity, equilibrium dissociation constant(K_(D)), dissociation and association rates (K_(off) and K_(on)respectively), binding affinity and/or avidity) and that certainalterations are more or less desirable, relevant to the application forthe antibodies. It is well known in the art that the equilibriumdissociation constant (K_(D)) is defined as k_(off)/k_(on). It isgenerally understood that a binding molecule (e.g., an antibody) with alow K_(D) is preferable to a binding molecule (e.g., an antibody) with ahigh K_(D). However, in some instances the value of the k_(on) ork_(off) may be more relevant than the value of the K_(D). One skilled inthe art can determine which kinetic parameter is most important for agiven antibody application. For example a modification that enhances Fcbinding to one or more positive regulators (e.g., FcγRIIIA) whileleaving unchanged or even reducing Fc binding to the negative regulatorFcγRIIB should correlate with enhanced ADCC activity. Alternatively, amodification that reduced binding to one or more positive regulatorand/or enhanced binding to FcγRIIB should correlate with reduced ADCCactivity. Accordingly, the ratio of binding affinities (e.g.,equilibrium dissociation constants (K_(D))) can indicate if the ADCCactivity of an Fc variant is enhanced or decreased. For example adecrease in the ratio of FcγRIIIA/FcγRIIB equilibrium dissociationconstants (K_(D)), should correlate with improved ADCC activity, whilean increase in the ratio should correlate with a decrease in ADCCactivity. Additionally, modifications that enhance binding to C1q shouldcorrelate with enhanced CDC activity, while modifications that reducebinding to C1q should correlate with reduced or eliminated CDC activity.

In one embodiment of the invention, the cell displayed Fc variantlibrary is screened for altered binding affinity for at least one Fcreceptor/ligand (e.g., FcγRIIIA, FcγRIIB, C1q, etc.) relative to apolypeptide having the same amino acid sequence as the Fc variant exceptcomprising an unmodified Fc region (referred to herein as a “comparablemolecule”). Accordingly, the present invention also provides Fc variants(also referred to herein as “Fc variants of the invention”) having analtered binding affinity for at least one Fc receptor/ligand (e.g.,FcγRIIIA, FcγRIIB, C1q, etc.) relative to a comparable molecule.

There are various methods known in the art that could be used to screenfor altered FcγR and/or C1q binding properties. In one embodiment, theFcγR (e.g. FcγRIIIA and/or FcγRIIB) and/or C1q to be used for screeningis fluorescently labeled, for example using techniques well known in theart including but not limited to those described herein (see e.g.,section entitled “Examples” infra). In one embodiment, the FcγR (e.g.FcγRIIIA and/or FcγRIIB) and/or C1q to be used for screening are afusion with streptavidin and labeled biotin is used for labeling cellsexpressing Fc. The labels can be detected as described herein. In oneembodiment, flow cytometry is used to sort/select antibody Fc regionswith the desired characteristics. For example, fluorescently labeledFcγRIIIA can be used to screen for Fc regions of antibodies with alteredbinding affinities. For example, the cells can be sorted based on meanfluorescent, with cells with high or increased mean fluorescent beingsorted as possible increased binding affinity or with cells with low ordecreased mean fluorescent being sorted as possible decreased bindingaffinity. In one embodiment, another labeled antibody that binds all ofthe variant Fc antibodies (e.g., an anti-human IgG antibody) can also beused to normalize for total antibody Fc displayed on the cell surface.For example, the cells can first be sorted for cells bound by ananti-human IgG antibody and then sorted for cells that bind (positiveselection) or those that do not bind (negative selection) FcγRIIIA. Inone embodiment, the cells can first be sorted for cells that bind(positive selection) or those that do not bind (negative selection)FcγRIIIA and then sorted for cells that bind an anti-human IgG antibody.In another embodiment, the cells that bind FcγRIIIA are labeled with afirst fluorescent molecule and the cells that bind an anti-human IgGantibody are labeled with a second fluorescent antibody, wherein thecells are simultaneously sorted for those positive for binding bothFcγRIIIA and the anti-human IgG antibody. In an alternative embodiment,the cells are simultaneously sorted for those negative (or with lowbinding affinity) for binding FcγRIIIA and positive for binding theanti-human IgG antibody.

In one embodiment of the invention, the cell displayed Fc variantlibrary is screened for increased or high binding to FcγRIIIA. In oneembodiment of the invention, the cell displayed Fc variant library isscreened for decreased or low binding affinity to FcγRIIIA. In oneembodiment, an Fc variant library of the invention is screened for anincreased or high affinity for FcγRIIIA and an affinity for FcγRIIB thatis unchanged, reduced or enhanced. In one embodiment, an Fc variantlibrary of the invention is screened for 1) an increased or highaffinity for FcγRIIIA; 2) an affinity for FcγRIIB that is unchanged,reduced, or increased and 3) an affinity for C1q that is unchanged,reduced, or increased. In another embodiment, an Fc variant library ofthe invention is screened for 1) a decreased affinity for FcγRIIIA; and2) an affinity for FcγRIIB that is increased.

In one embodiment, an Fc variant library of the invention is screenedfor a ratio of FcγRIIIA/FcγRIIB equilibrium dissociation constants (KD)that is increased or high. In another embodiment, an Fc variant libraryof the invention is screened for ratio of FcγRIIIA/FcγRIIB equilibriumdissociation constants (KD), is decreased or low. In one embodiment ofthe invention, the cell displayed Fc variant library is screened forincreased or high binding for FcγRIIB. In one embodiment of theinvention, the cell displayed Fc variant library is screened fordecreased or low binding for FcγRIIB. In one embodiment of theinvention, the cell displayed Fc variant library is screened fordecreased or low binding affinity for C1q. In one embodiment of theinvention, the cell displayed Fc variant library is screened forincreased or high binding affinity for C1q.

In one embodiment, an Fc variant of the invention has increased or highbinding affinity to FcγRIIIA. In another embodiment of the invention, anFc variant of the invention has decreased or low binding affinity toFcγRIIIA. In still another embodiment, an Fc variant of the inventionhas increased or high affinity for FcγRIIIA and an affinity for FcγRIIBthat is unchanged, reduced or enhanced. In yet another embodiment, an Fcvariant of the invention has 1) an increased or high affinity forFcγRIIIA; 2) an affinity for FcγRIIB that is unchanged, reduced, orincreased and 3) an affinity for C1q that is unchanged, reduced, orincreased. In another embodiments, an Fc variant of the invention has 1)a decreased affinity for FcγRIIIA; and 2) an affinity for FcγRIIB thatis increased. In certain embodiments, the binding affinity of an Fcvariant of the invention to FcγRIIIA and/or FcγRIIB and/or C1q isincreased or decreased relative to a comparable molecule.

In one embodiment, an Fc variant of the invention has a ratio ofFcγRIIIA/FcγRIIB equilibrium dissociation constants (KD) that isincreased or high. In another embodiment, an Fc variant of the inventionhas ratio of FcγRIIIA/FcγRIIB equilibrium dissociation constants (KD),is decreased or low. In still another embodiment of the invention, an Fcvariant of the invention has increased or high binding for FcγRIIB. Inone embodiment of the invention, the Fc variant of the invention hasdecreased or low binding for FcγRIIB. In other embodiments of theinvention, an Fc variant of the invention has decreased or low bindingaffinity for C1q. In still other embodiments of the invention, an Fcvariant of the invention has increased or high binding affinity for C1q.In certain embodiments, the binding affinity of an Fc variant of theinvention to FcγRIIIA and/or FcγRIIB and/or C1q is increased ordecreased relative to a comparable molecule.

In a specific embodiment, an Fc variant of the invention has an affinityfor an Fc receptor and/or ligand (e.g., FcγRIIIA, C1q) that is at least2 fold, or at least 3 fold, or at least 5 fold, or at least 7 fold, or aleast 10 fold, or at least 20 fold, or at least 30 fold, or at least 40fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, orat least 80 fold, or at least 90 fold, or at least 100 fold, or at least200 fold greater than that of a comparable molecule. In otherembodiments, an Fc variant of the invention has an affinity for an Fcreceptor and/or ligand (e.g., FcγRIIIA, C1q) that is increased by atleast 10%, or at least 20%, or at least 30%, or at least 40%, or atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 100%, or at least 150%, or at least 200%,relative to a comparable molecule.

In a specific embodiment, an Fc variant of the invention has anequilibrium dissociation constant (K_(D)) for an Fc receptor and/orligand (e.g., FcγRIIIA, C1q) that is reduced by at least 2 fold, or atleast 3 fold, or at least 5 fold, or at least 7 fold, or a least 10fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, orat least 50 fold, or at least 60 fold, or at least 70 fold, or at least80 fold, or at least 90 fold, or at least 100 fold, or at least 200fold, or at least 400 fold, or at least 600 fold, relative to acomparable molecule. In another specific embodiment, an Fc variant ofthe invention has an equilibrium dissociation constant (K_(D)) for an Fcreceptor and/or ligand (e.g., FcγRIIIA, C1q) that is reduced by at least10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%,or at least 60%, or at least 70%, or at least 80%, or at least 90%, orat least 100%, or at least 150%, or at least 200%, relative to acomparable molecule.

In a specific embodiment, an Fc variant of the invention has an affinityfor an Fc receptor and/or ligand (e.g., FcγRIIIA, C1q) that is reducedby at least 2 fold, or at least 3 fold, or at least 5 fold, or at least7 fold, or a least 10 fold, or at least 20 fold, or at least 30 fold, orat least 40 fold, or at least 50 fold, or at least 60 fold, or at least70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold,or at least 200 fold, relative to a comparable molecule. In otherembodiments, an Fc variant of the invention has an affinity for an Fcreceptor and/or ligand (e.g., FcγRIIIA, C1q) that is decreased by atleast 10%, or at least 20%, or at least 30%, or at least 40%, or atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 100%, or at least 150%, or at least 200%,relative to a comparable molecule.

In a specific embodiment, an Fc variant of the invention has anequilibrium dissociation constant (K_(D)) for an Fc receptor and/orligand (e.g., FcγRIIIA, C1q) that is increased by at least 2 fold, or atleast 3 fold, or at least 5 fold, or at least 7 fold, or a least 10fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, orat least 50 fold, or at least 60 fold, or at least 70 fold, or at least80 fold, or at least 90 fold, or at least 100 fold, or at least 200fold, or at least 400 fold, or at least 600 fold, relative to acomparable molecule. In another specific embodiment, an Fc variant ofthe invention has an equilibrium dissociation constant (K_(D)) for an Fcreceptor and/or ligand (e.g., FcγRIIIA, C1q) that is increased by atleast 10%, or at least 20%, or at least 30%, or at least 40%, or atleast 50%, or at least 60%, or at least 70%, or at least 80%, or atleast 90%, or at least 100%, or at least 150%, or at least 200%,relative to a comparable molecule.

In some embodiments, the Fc variant antibody library is screened forincreased or decreased ADCC and/or CDC relative to a comparablemolecule. Accordingly, the present invention also provides Fc variantshaving increased or decreased ADCC and/or CDC relative to a comparablemolecule.

In one embodiment of the invention, the cell displayed Fc variantlibrary is screened for high, low, increase, decreased or essentiallyunchanged ADCC activity. In a specific embodiment, the cell displayed Fcvariant library is screened for high or increased ADCC activity. Inanother specific embodiment, the cell displayed Fc variant library isscreened for low or decreased ADCC activity. In still another specificembodiment, the cell displayed Fc variant library is screened forunchanged ADCC activity. In one embodiment of the invention, the celldisplayed Fc variant library is screened for high, low, increase,decreased or essentially unchanged CDC activity. In a specificembodiment, the cell displayed Fc variant library is screened for highor increased CDC activity. In another specific embodiment, the celldisplayed Fc variant library is screened for low or decreased CDCactivity. In still another specific embodiment, the cell displayed Fcvariant library is screened for unchanged CDC activity.

In one embodiment, an Fc variant of the invention has high or increasedADCC activity. In another embodiment, an Fc variant of the invention haslow or decreased ADCC activity. In still another embodiment, an Fcvariant of the invention has unchanged ADCC activity. In otherembodiments, an Fc variant of the invention has high or increased CDCactivity. In still other embodiments, an Fc variant of the invention haslow or decreased CDC activity. In yet other embodiments, an Fc variantof the invention has unchanged CDC activity. In certain embodiments,ADCC and/or CDC of an Fc variant of the invention is increased,decreased or unchanged relative to a comparable molecule.

In a specific embodiment, an Fc variant of the invention has ADCC and/orCDC activity that is at least 2 fold, or at least 3 fold, or at least 5fold, or at least 10 fold, or at least 50 fold, or at least 100 foldgreater than that of a comparable molecule. In yet another embodiment,an Fc variant of the invention has ADCC and/or CDC activity that isincreased by at least 10%, or at least 20%, or at least 30%, or at least40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%,or at least 90%, or at least 100%, or at least 150%, or at least 200%,relative to a comparable molecule.

In another specific embodiment, an Fc variant of the invention has ADCCand/or CDC activity that is reduced by at least 2 fold, or at least 3fold, or at least 5 fold, or at least 10 fold, or at least 50 fold, orat least 100 fold, relative to a comparable molecule. In yet anotherembodiment, an Fc variant of the invention has ADCC and/or CDC activitythat is decreased by at least 10%, or at least 20%, or at least 30%, orat least 40%, or at least 50%, or at least 60%, or at least 70%, or atleast 80%, or at least 90%, or at least 100%, or at least 150%, or atleast 200%, relative to a comparable molecule.

An Fc variant of the invention comprises an Fc region with at least oneamino acid substitution, deletion or insertion, referred to herein as a“variant Fc region of the invention”. It is contemplated that a variantFc region of the invention can be incorporated into additionalmolecules, such as Fc fusion proteins or other antibodies, to modulateFc receptor (e.g., FcγR) and/or Fc ligand (e.g., C1q) binding and/oreffector function (e.g., ADCC activity). This may be accomplished “denovo” by combining a heterologous molecule with the variant Fc region ofthe invention. Alternatively, or optionally, this may be accomplished bymodifying the Fc region of an Fc region-containing polypeptide tocomprises the same amino acid substitution, deletion or insertionpresent in the variant Fc region of the invention. Accordingly, thepresent invention provides methods to modulate Fc receptor (e.g., FcγR)and/or Fc ligand (e.g., C1q) binding and/or effector function (e.g.,ADCC activity) comprising introducing a variant Fc region of theinvention into an Fc containing polypeptide. Methods for generatingfusion proteins and introducing amino acid substitutions, deletions orinsertions are well known in the art and include chemical synthesis andrecombinant expression techniques. (see, e.g., Current Protocols inMolecular Biology, F. M. Ausubel et al., eds., John Wiley & Sons (NY,1998); Molecular Cloning: A Laboratory Manual, 3nd Edition, J. Sambrooket al., eds., Cold Spring Harbor Laboratory Press (NY, 2001)).

In one embodiment, the present invention provides a method of generatingan Fc variant with altered Fc receptor (e.g., FcγR) and/or Fc ligand(e.g., C1q) binding and/or effector function (e.g., ADCC activity)comprising introducing at least one amino acid residue substitution(i.e., a non naturally occurring amino acid residue), deletion orinsertion at a position selected from the group consisting of amino acidresidues: 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 242,246, 250, 251, 256, 257, 259, 260, 261, 265, 266, 269, 273, 274, 275,277, 281, 282, 298, 327, 328, 329, 330, 332, 346, 347 and 348, whereinthe numbering system is that of the EU index as set forth in Kabat.

In one embodiment, the present invention provides a method of generatingan Fc variant with altered Fc receptor (e.g., FcγR) and/or Fc ligand(e.g., C1q) binding and/or effector function (e.g., ADCC activity)comprising introducing at least one amino acid residue substitution(i.e., a non naturally occurring amino acid residue), deletion orinsertion at a position selected from the group consisting of amino acidresidues: 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 242,246, 250, 251, 256, 257, 259, 260, 261, 265, 266, 269, 273, 274, 275,277, 281, 282, 298, 327, 328, 329, 330, 332, 346 and 348, wherein thenumbering system is that of the EU index as set forth in Kabat.

In another embodiment, the present invention provides a method ofgenerating an Fc variant with altered Fc receptor and/or Fc ligandbinding and/or effector function comprising introducing at least one nonnaturally occurring amino acid residue selected from the groupconsisting of: 231L, 231I, 231V, 231N, 231Q, 231T, 231S, 232K, 232R,234K, 234R, 235V, 235I, 235A, 235G, 236K, 236R, 236L, 236I, 236V, 236A,237R, 237K, 238N, 238Q, 238V, 238L, 238I, 238E, 238D, 238A, 238G, 238M,238C, 239D, 240G, 240A, 240H, 240D, 240E, 246R, 246E, 246D, 246W, 246F,246M, 246C, 250S, 250V, 250I, 250L, 251A, 251G, 251E, 251D, 251V, 251I,256R, 256K, 260R, 260K, 260E, 260D, 261S, 261T, 266A, 266G, 274R, 277V,277I, 277L, 277S, 277T, 281S, 28 1T, 282F, 282W, 346R, 346K, 348G and348A, wherein the numbering system is that of the EU index as set forthin Kabat.

In a specific embodiment, the present invention provides a method ofgenerating an Fc variant with altered Fc receptor and/or Fc ligandbinding and/or effector function comprising introducing at least one nonnaturally occurring amino acid residues selected from the groupconsisting of: 231L, 231N, 231T, 232K, 234R, 235V, 235A, 235I, 236R,236V, 236A, 237R, 237G 238N, 238V, 238E, 238L, 238G, 238M, 238Q, 239D,240G, 240H, 240E, 246R, 246E, 246W, 246M, 250S, 250V, 251A, 251E, 251I,256R, 260R, 260E, 261S, 265, 266A, 274R, 277V, 277T, 281S, 282F, 346Rand 348A, wherein the numbering system is that of the EU index as setforth in Kabat.

In another specific embodiment, the present invention provides a methodof generating an Fc variant with altered Fc receptor and/or Fc ligandbinding and/or effector function comprising introducing at least one nonnaturally occurring amino acid residues selected from the groupconsisting of: 198T, 234R, 236R, 236A, 237R, 238L, 238E, 238N, 238V,238Q, 240E, 240G, 248E, 251A, 251E, 266A, 277T. In yet another specificembodiment, an Fc variant of the present invention comprises at leastone combination of non naturally occurring amino acid residues selectedfrom the group consisting of: 246R/251E/260R, 240G/198T, 237R/236A.

In certain embodiments, the present invention provides a method ofgenerating an Fc variant with altered Fc receptor and/or Fc ligandbinding and/or effector function comprising introducing at least oneinsertion after a position selected from the group consisting of aminoacid residues: 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240,242, 246, 250, 251, 256, 257, 259, 260, 261, 265, 266, 269, 273, 274,275, 277, 281, 282, 298, 327, 328, 329, 330, 332, 346 and 348, whereinthe numbering system is that of the EU index as set forth in Kabat. Theinsertion may be any amino acid residue. Specific insertions may beidentified herein as “In” followed by the one letter code of theinserted amino acid residue and the position of the residues immediatelyflanking the insertion. For example “InG231/232” denotes a variant Fccomprising an insertion of a glycine between residues 231 and 232. Incertain embodiments an Fc variant of the invention may comprises theinsertion of more than one amino acid residue after a selected position.In certain other embodiments, an Fc variant of the invention maycomprises the insertion of one or more amino acid residues aftermultiple positions.

In a specific embodiment, the present invention provides a method ofgenerating an Fc variant with altered Fc receptor and/or Fc ligandbinding and/or effector function comprising introducing at least oneinsertion after a position selected from the group consisting of aminoacid residues: 230, 231, 232, 233, 234, 235, 236, 237, 238, 239 and 240,wherein the numbering system is that of the EU index as set forth inKabat. The insertion may be any amino acid residue. In certainembodiments an Fc variant of the invention may comprises the insertionof more than one amino acid residue after a selected position. Incertain other embodiments, an Fc variant of the invention may comprisesthe insertion of one or more amino acid residues after multiplepositions.

In another specific embodiment, the present invention provides a methodof generating an Fc variant with altered Fc receptor and/or Fc ligandbinding and/or effector function comprising introducing at least oneinsertion selected from the group consisting of: InR234/235; InV235/236;InR236/237; InR237/238; InV238/239; InN238/239; InL238/239; InE238/239;InG238/239; InS239/240; InG240/241 and InE240/241.

In certain embodiments, the present invention provides a method ofgenerating an Fc variant with altered Fc receptor and/or Fc ligandbinding and/or effector function comprising introducing a combination asubstitution and an insertion. In other embodiments, the presentinvention provides a method of generating an Fc variant with altered Fcreceptor and/or Fc ligand binding and/or effector function comprisingintroducing combination of one or more of the substitutions and one ormore of the insertions disclosed herein. In a specific embodiment, thepresent invention provides a method of generating an Fc variant withaltered Fc receptor and/or Fc ligand binding and/or effector functioncomprising introducing at least one combinations of insertions andsubstitutions selected from the group consisting of: InG240/241/1198T,InL238/239/P238Q, InE238/239/V348A, InS239/240/V266A, andInR237/238/G236A.

7.6 Antibodies

Essentially all types of antibodies may be utilized in accordance withthe invention. These include, but are not limited to, syntheticantibodies, monoclonal antibodies, recombinantly produced antibodies,intrabodies, multispecific antibodies, diabodies, bispecific antibodies,human antibodies, humanized antibodies, chimeric antibodies, syntheticantibodies, single-chain Fvs (scFv), Fab fragments, F(ab′) fragments,disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies,and epitope-binding fragments of any of the above. Antibodies used inthe methods of the present invention include immunoglobulin moleculesand immunologically active portions of immunoglobulin molecules. Theimmunoglobulin molecules of the invention can be of any type (e.g., IgG,IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1and IgA2) or subclass of immunoglobulin molecule.

Antibodies or antibody fragments may be from any animal origin includingbirds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat,guinea pig, camel, horse, or chicken). In one embodiment, the antibodiesare human or humanized monoclonal antibodies. As used herein, “human”antibodies include antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from mice that express antibodies from human genes.Antibodies or antibody fragments used in accordance with the presentinvention may be monospecific, bispecific, trispecific or of greatermultispecificity. Multispecific antibodies may specifically bind todifferent epitopes of desired target molecule or may specifically bindto both the target molecule as well as a heterologous epitope, such as aheterologous polypeptide or solid support material. See, e.g., PCTPublication Nos. WO 93/17715, WO 92/08802, WO 91/00360, and WO 92/05793;Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893,4,714,681, 4,925,648, 5,573,920, and 5,601,819; and Kostelny et al.,1992, J. Immunol. 148:1547-1553. The present invention may also bepracticed with single domain antibodies, including camelized singledomain antibodies (see e.g., Muyldermans et al., 2001, Trends Biochem.Sci. 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1:253; Reichmannand Muyldermans, 1999, J. Immunol. Meth. 231:25; PCT Publication Nos. WO94/04678 and WO 94/25591; U.S. Pat. No. 6,005,079).

Embodiments of the invention include antibodies that bind to any target.Antibodies may be from any species, be chimeric antibodies or humanizedantibodies. In one embodiment, the antibodies are human antibodies. Inone embodiment, the antibodies are humanized antibodies.

It is also contemplated that an Fc variant library may be generatedfrom, or a variant Fc region of the invention may be introduced into anantibody already described in the art including but not limited toanti-fluorescein monoclonal antibody, 4-4-20 (Kranz et al., 1982 J.Biol. Chem. 257(12): 6987-6995), a humanized anti-TAG72 antibody (CC49)(Sha et al., 1994 Cancer Biother. 9(4): 341-9), an antibody thatspecifically bind an Eph Receptor including, but not limited to thosedisclosed in PCT Publication Nos. WO 04/014292, WO 03/094859 and U.S.patent application Ser. No. 10/863,729, antibodies that specificallybind Integrin α_(V)β₃ including, but not limited to, LM609 (Scripps),the murine monoclonal LM609 (PCT Publication WO 89/015155 and U.S. Pat.No. 5,753,230); the humanized monoclonal antibody MEDI-522 (a.k.a.VITAXIN®, MedImmune, Inc., Gaithersburg, Md.; Wu et al., 1998, PNAS USA95(11): 6037-6042; PCT Publications WO 90/33919 and WO 00/78815), anantibody against interferon alpha as disclosed in WO/2005/05059106, anantibody against the interferon receptor 1 as disclosed inWO/2006/059106, Erbitux™ (also known as IMC-C225) (Imclone SystemsInc.), a chimerized monoclonal antibody against EGFR; HERCEPTIN®(Trastuzumab) (Genentech, CA) which is a humanized anti-HER2 monoclonalantibody for the treatment of patients with metastatic breast cancer;REOPRO® (abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIareceptor on the platelets for the prevention of clot formation; ZENAPAX®(daclizumab) (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection. Other examples are ahumanized anti-CD18 F(ab′)₂ (Genentech); CDP860 which is a humanizedanti-CD18 F(ab′)₂ (Celltech, UK); PRO542 which is an anti-HIV gp120antibody fused with CD4 (Progenics/Genzyme Transgenics); C14 which is ananti-CD14 antibody (ICOS Pharm); a humanized anti-VEGF IgG1antibody(Genentech); OVAREX™ which is a murine anti-CA 125 antibody (Altarex);PANOREX™ which is a murine anti-17-IA cell surface antigen IgG2aantibody (Glaxo Wellcome/Centocor); IMC-C225 which is a chimericanti-EGFR IgG antibody (Imclone System); VITAXIN™ which is a humanizedanti-αVβ3 integrin antibody (Applied Molecular Evolution/MedImmune);Campath 1H/LDP-03 which is a humanized anti CD52 IgG1 antibody(Leukosite); Smart M195 which is a humanized anti-CD33 IgG antibody(Protein Design Lab/Kanebo); RITUXAN™ which is a chimeric anti-CD20 IgG1antibody (IDEC Pharm/Genentech, Roche/Zettyaku); LYMPHOCIDE™ which is ahumanized anti-CD22 IgG antibody (Immunomedics); Smart ID10 which is ahumanized anti-HLA antibody (Protein Design Lab); ONCOLYM™ (Lym-1) is aradiolabelled murine anti-HLA DR antibody (Techniclone); anti-CD11a is ahumanized IgG1 antibody (Genetech/Xoma); ICM3 is a humanized anti-ICAM3antibody (ICOS Pharm); IDEC-114 is a primatized anti-CD80 antibody (IDECPharm/Mitsubishi); ZEVALIN™ is a radiolabelled murine anti-CD20 antibody(IDEC/Schering AG); IDEC-131 is a humanized anti-CD40L antibody(IDEC/Eisai); IDEC-151 is a primatized anti-CD4 antibody (IDEC);IDEC-152 is a primatized anti-CD23 antibody (IDEC/Seikagaku); SMARTanti-CD3 is a humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is ahumanized anti-complement factor 5 (C5) antibody (Alexion Pharm);IDEC-151 is a primatized anti-CD4 IgG1 antibody (IDEC Pharm/SmithKlineBeecham); MDX-CD4 is a human anti-CD4 IgG antibody(Medarex/Eisai/Genmab); CDP571 is a humanized anti-TNF-α IgG4 antibody(Celltech); LDP-02 is a humanized anti-α4β7 antibody(LeukoSite/Genentech); Orthoclone OKT4A is a humanized anti-CD4 IgGantibody (Ortho Biotech); ANTOVA™ is a humanized anti-CD40L IgG antibody(Biogen); ANTEGREN™ is a humanized anti-VLA-4 IgG antibody (Elan);MDX-33 is a human anti-CD64 (FcγR) antibody (Medarex/Centeon);rhuMab-E25 is a humanized anti-IgE IgG1 antibody(Genentech/Norvartis/Tanox Biosystems); IDEC-152 is a primatizedanti-CD23 antibody (IDEC Pharm); ABX-CBL is a murine anti CD-147 IgMantibody (Abgenix); BTI-322 is a rat anti-CD2 IgG antibody(Medimmune/Bio Transplant); Orthoclone/OKT3 is a murine anti-CD3 IgG2aantibody (ortho Biotech); SIMULECT™ is a chimeric anti-CD25 IgG1antibody (Novartis Pharm); LDP-01 is a humanized anti-β₂-integrin IgGantibody (LeukoSite); Anti-LFA-1 is a murine anti CD18 F(ab′)₂(Pasteur-Merieux/Immunotech); CAT-152 is a human anti-TGF-β₂ antibody(Cambridge Ab Tech); and Corsevin M is a chimeric anti-Factor VIIantibody (Centocor).

Additional antibodies which may be utilized in accordance with thepresent invention may specifically bind a cancer or tumor antigen forexample, including, but not limited to, KS 1/4 pan-carcinoma antigen(Perez and Walker, 1990, J. Immunol. 142: 3662-3667; Bumal, 1988,Hybridoma 7(4): 407-415), ovarian carcinoma antigen (CA125) (Yu et al.,1991, Cancer Res. 51(2): 468-475), prostatic acid phosphate (Tailor etal., 1990, Nucl. Acids Res. 18(16): 4928), prostate specific antigen(Henttu and Vihko, 1989, Biochem. Biophys. Res. Comm. 160(2): 903-910;Israeli et al., 1993, Cancer Res. 53: 227-230), melanoma-associatedantigen p97 (Estin et al., 1989, J. Natl. Cancer Instit. 81(6):445-446), melanoma antigen gp75 (Vijayasardahl et al., 1990, J. Exp.Med. 171(4): 1375-1380), high molecular weight melanoma antigen(HMW-MAA) (Natali et al., 1987, Cancer 59: 55-63; Mittelman et al.,1990, J. clin. Invest. 86: 2136-2144), prostate specific membraneantigen, carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am.Soc. clin. Oncol. 13: 294), polymorphic epithelial mucin antigen, humanmilk fat globule antigen, colorectal tumor-associated antigens such as:CEA, TAG-72 (Yokata et al., 1992, Cancer Res. 52: 3402-3408), CO17-1A(Ragnhammar et al., 1993, Int. J. Cancer 53: 751-758); GICA 19-9 (Herlynet al., 1982, J. clin. Immunol. 2: 135), CTA-1 and LEA, Burkitt'slymphoma antigen-38.13, CD19 (Ghetie et al., 1994, Blood 83: 1329-1336),human B-lymphoma antigen-CD20 (Reff et al., 1994, Blood 83:435-445),CD33 (Sgouros et al., 1993, J. Nucl. Med. 34:422-430), melanoma specificantigens such as ganglioside GD2 (Saleh et al., 1993, J. Immunol., 151,3390-3398), ganglioside GD3 (Shitara et al., 1993, Cancer Immunol.Immunother. 36:373-380), ganglioside GM2 (Livingston et al., 1994, J.clin. Oncol. 12: 1036-1044), ganglioside GM3 (Hoon et al., 1993, CancerRes. 53: 5244-5250), tumor-specific transplantation type of cell-surfaceantigen (TSTA) such as virally-induced tumor antigens includingT-antigen DNA tumor viruses and Envelope antigens of RNA tumor viruses,oncofetal antigen-alpha-fetoprotein such as CEA of colon, bladder tumoroncofetal antigen (Hellstrom et al., 1985, Cancer. Res. 45:2210-2188),differentiation antigen such as human lung carcinoma antigen L6, L20(Hellstrom et al., 1986, Cancer Res. 46: 3917-3923), antigens offibrosarcoma, human leukemia T cell antigen-Gp37(Bhattacharya-Chatterjee et al., 1988, J. of Immun. 141:1398-1403),neoglycoprotein, sphingolipids, breast cancer antigen such as EGFR(Epidermal growth factor receptor), HER2 antigen (p185^(HER2)),polymorphic epithelial mucin (PEM) (Hilkens et al., 1992, Trends in Bio.Chem. Sci. 17:359), malignant human lymphocyte antigen-APO-1 (Bernhardet al., 1989, Science 245: 301-304), differentiation antigen (Feizi,1985, Nature 314: 53-57) such as I antigen found in fetal erythrocytes,primary endoderm I antigen found in adult erythrocytes, preimplantationembryos, I(Ma) found in gastric adenocarcinomas, M18, M39 found inbreast epithelium, SSEA-1 found in myeloid cells, VEP8, VEP9, Myl,VIM-D5, D₁56-22 found in colorectal cancer, TRA-1-85 (blood group H),C14 found in colonic adenocarcinoma, F3 found in lung adenocarcinoma,AH6 found in gastric cancer, Y hapten, Le^(y) found in embryonalcarcinoma cells, TL5 (blood group A), EGF receptor found in A431 cells,E1 series (blood group B) found in pancreatic cancer, FC10.2 found inembryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514 (bloodgroup Le^(a)) found in Adenocarcinoma, NS-10 found in adenocarcinomas,CO-43 (blood group Le^(b)), G49 found in EGF receptor of A431 cells, MH2(blood group ALe^(b)/Le^(y)) found in colonic adenocarcinoma, 19.9 foundin colon cancer, gastric cancer mucins, T₅A₇ found in myeloid cells, R₂₄found in melanoma, 4.2, G_(D3), D1.1, OFA-1, G_(M2), OFA-2, G_(D2), andM1:22:25:8 found in embryonal carcinoma cells, and SSEA-3 and SSEA-4found in 4 to 8-cell stage embryos. In one embodiment, the antigen is aT cell receptor derived peptide from a Cutaneous Tcell Lymphoma (see,Edelson, 1998, The Cancer Journal 4:62).

7.7 Specific Antigens and Fusion Partners of the Invention

As described above, the methods of the present invention may be appliedto any antibody. For example an Fc variant library may be generatedfrom, or a variant Fc region of the invention may be introduced into anyantibody. Furthermore, an variant Fc region of the invention may beutilized to generate an Fc fusion protein. Accordingly, virtually anymolecule may be targeted by and/or incorporated into an antibody and/orFc fusion protein which may be utilized in accordance with the presentinvention including, but not limited to, the following list of proteins,as well as subunits, domains, motifs and epitopes belonging to thefollowing list of proteins: renin; a growth hormone, including humangrowth hormone and bovine growth hormone; growth hormone releasingfactor; parathyroid hormone; thyroid stimulating hormone; lipoproteins;alpha-1-antitrypsin; insulin A-chain; insulin B-chain; proinsulin;follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon;clotting factors such as factor VII, factor VIIIC, factor IX, tissuefactor (TF), and von Willebrands factor; anti-clotting factors such asProtein C; atrial natriuretic factor; lung surfactant; a plasminogenactivator, such as urokinase or human urine or tissue-type plasminogenactivator (t-PA); bombesin; thrombin; hemopoietic growth factor; tumornecrosis factor-alpha and -beta; enkephalinase; RANTES (regulated onactivation normally T-cell expressed and secreted); human macrophageinflammatory protein (MIP-1-alpha); a serum albumin such as human serumalbumin; Muellerian-inhibiting substance; relaxin A-chain; relaxinB-chain; prorelaxin; mouse gonadotropin-associated peptide; a microbialprotein, such as beta-lactamase; DNase; IgE; a cytotoxic T-lymphocyteassociated antigen (CTLA), such as CTLA-4; inhibin; activin; vascularendothelial growth factor (VEGF); receptors for hormones or growthfactors such as, for example, EGFR, VEGFR; interferons such as alphainterferon (α-IFN), beta interferon (β-IFN) and gamma interferon(γ-IFN); interferon receptor components such as interferon receptor 1;protein A or D; rheumatoid factors; a neurotrophic factor such asbone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6(NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor; platelet-derivedgrowth factor (PDGF); fibroblast growth factor such as αFGF and βFGF;epidermal growth factor (EGF); transforming growth factor (TGF) such asTGF-alpha and TGF-beta, including TGF-1, TGF-2, TGF-3, TGF-4, or TGF-5;insulin-like growth factor-I and -II (IGF-I and IGF-II); des (1-3)-IGF-I(brain IGF-I), insulin-1ike growth factor binding proteins; CD proteinssuch as CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD19, CD20, CD22, CD23,CD25, CD33, CD34, CD40, CD40L, CD52, CD63, CD64, CD80 and CD147;erythropoietin; osteoinductive factors; immunotoxins; a bonemorphogenetic protein (BMP); an interferon such asinterferon-alpha,-beta, and-gamma; colony stimulating factors (CSFs),such as M-CSF, GM-CSF, and G-CSF; interleukins (ILs), e.g., IL-1 toIL-13; TNFα, HMGB1; HMGB2; superoxide dismutase; T-cell receptors;surface membrane proteins; decay accelerating factor; viral antigen suchas, for example, a portion of the AIDS envelope, e.g.,gp120; transportproteins; homing receptors; addressins; regulatory proteins; celladhesion molecules such as LFA-1, Mac 1, p150.95, VLA-4, ICAM-1, ICAM-3and VCAM, a4/p7 integrin, and (Xv/p3 integrin including either a orsubunits thereof, integrin alpha subunits such as CD49a, CD49b, CD49c,CD49d, CD49e, CD49f, alpha7, alpha8, alpha9, alphaD, CD11a, CD11b, CD51,CD11c, CD41, alphaIIb, alphaIELb; integrin beta subunits such as, CD29,CD 18, CD61, CD104, beta5, beta6, beta7 and beta8; Integrin subunitcombinations including but not limited to, αVβ3, αVβ5 and α4β7; a memberof an apoptosis pathway; IgE; blood group antigens; flk2/flt3 receptor;obesity (OB) receptor; mp1 receptor; CTLA-4; protein C; a chitinase orchitinase-like molecule such as YKL-40 and AMCase; an Eph receptor suchas EphA2, EphA4, EphB2, etc.; a Human Leukocyte Antigen (HLA) such asHLA-DR; complement proteins such as complement receptor CR1, C1Rq andother complement factors such as C3, and C5; a glycoprotein receptorsuch as GpIba, GPIIb/IIIa and CD200;

Additional, molecules which may be utilized in accordance with thepresent invention are those that specifically bind cancer antigensincluding, but not limited to, ALK receptor (pleiotrophin receptor),pleiotrophin, KS 1/4 pan-carcinoma antigen; ovarian carcinoma antigen(CA125); prostatic acid phosphate; prostate specific antigen (PSA);melanoma-associated antigen p97; melanoma antigen gp75; high molecularweight melanoma antigen (HMW-MAA); prostate specific membrane antigen;carcinoembryonic antigen (CEA); polymorphic epithelial mucin antigen;human milk fat globule antigen; colorectal tumor-associated antigenssuch as: CEA, TAG-72, CO17-1A, GICA 19-9, CTA-1 and LEA; Burkitt'slymphoma antigen-38.13; CD19; human B-lymphoma antigen-CD20; CD33;melanoma specific antigens such as ganglioside GD2, ganglioside GD3,ganglioside GM2 and ganglioside GM3; tumor-specific transplantation typecell-surface antigen (TSTA); virally-induced tumor antigens includingT-antigen, DNA tumor viruses and Envelope antigens of RNA tumor viruses;oncofetal antigen-alpha-fetoprotein such as CEA of colon, 5T4 oncofetaltrophoblast glycoprotein and bladder tumor oncofetal antigen;differentiation antigen such as human lung carcinoma antigens L6 andL20; antigens of fibrosarcoma; human leukemia T cell antigen-Gp37;neoglycoprotein; sphingolipids; breast cancer antigens such as EGFR(Epidermal growth factor receptor); NY-BR-16; NY-BR-16 and HER2 antigen(p185^(HER2)); polymorphic epithelial mucin (PEM); malignant humanlymphocyte antigen-APO-1; differentiation antigen such as I antigenfound in fetal erythrocytes; primary endoderm I antigen found in adulterythrocytes; preimplantation embryos; I(Ma) found in gastricadenocarcinomas; M18, M39 found in breast epithelium; SSEA-1 found inmyeloid cells; VEP8; VEP9; Myl; VIM-D5; D₁56-22 found in colorectalcancer; TRA-1-85 (blood group H); SCP-1 found in testis and ovariancancer; C14 found in colonic adenocarcinoma; F3 found in lungadenocarcinoma; AH6 found in gastric cancer; Y hapten; Le^(y) found inembryonal carcinoma cells; TL5 (blood group A); EGF receptor found inA431 cells; E₁ series (blood group B) found in pancreatic cancer; FC10.2found in embryonal carcinoma cells; gastric adenocarcinoma antigen;CO-514 (blood group Le^(a)) found in Adenocarcinoma; NS-10 found inadenocarcinomas; CO-43 (blood group Le^(b)); G49 found in EGF receptorof A431 cells; MH2 (blood group ALe^(b)/Le^(y)) found in colonicadenocarcinoma; 19.9 found in colon cancer; gastric cancer mucins; T₅A₇found in myeloid cells; R₂₄ found in melanoma; 4.2, G_(D3), D1.1, OFA-1,G_(M2), OFA-2, G_(D2), and M11:22:25:8 found in embryonal carcinomacells and SSEA-3 and SSEA-4 found in 4 to 8-cell stage embryos;Cutaneous Tcell Lymphoma antigen; MART-1 antigen; Sialy Tn (STn)antigen; Colon cancer antigen NY-CO-45; Lung cancer antigen NY-LU-12variant A; Adenocarcinoma antigen ART1; Paraneoplastic associatedbrain-testis-cancer antigen (onconeuronal antigen MA2; paraneoplasticneuronal antigen); Neuro-oncological ventral antigen 2 (NOVA2);Hepatocellular carcinoma antigen gene 520; TUMOR-ASSOCIATED ANTIGENCO-029; Tumor-associated antigens MAGE-C1 (cancer/testis antigen CT7),MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM6), MAGE-2, MAGE-4a, MAGE-4b andMAGE-X2; Cancer-Testis Antigen (NY-EOS-1); YKL-40 and fragments of anyof the above-listed polypeptides.

7.8 Downstream Engineering

It is contemplated that one or more of the polypeptides isolated usingthe screening methods of the present invention may be further modified.For example, an antibody isolated in accordance with the presentinvention may be modified (i.e., by the covalent attachment of any typeof molecule to the antibody such that covalent attachment). For example,but not by way of limitation, the antibody derivatives includeantibodies that have been modified, e.g., by glycosylation, acetylation,pegylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to a cellularligand or other protein, etc. Any of numerous chemical modifications maybe carried out by known techniques, including, but not limited to,specific chemical cleavage, acetylation, formylation, etc. In certainembodiments antibodies, or fragments thereof, isolated in accordancewith the present invention are fused to a bioactive molecule including,but not limited to, peptides, polypeptides, proteins, small molecules,mimetic agents, synthetic drugs, inorganic molecules, and organicmolecules. In other embodiments antibodies, or fragments thereof,isolated in accordance with the present invention are conjugated to adiagnostic, detectable or therapeutic agent. Such agents and method forconjugation are well known to one of skill in the art and are disclosedin numerous sources (see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119; International Publication Nos. WO 93/15199; WO93/15200; WO 97/33899; WO 97/34911; WO 01/77137; WO 03/075957; U.S.Patent Publications 2006/0040325).

Alternatively or optionally, the antibody, or a fragment thereof,isolated in accordance with the present invention may be fused to apolypeptide moiety. Methods for fusing or conjugating antibodies topolypeptide moieties are known in the art. See, e.g., U.S. Pat. Nos.5,336,603; 5,622,929; 5,359,046; 5,349,053; 5,447,851, and 5,112,946; EP307,434; EP 367,166; PCT Publications WO 96/04388 and WO 91/06570;Ashkenazi et al., 1991, PNAS USA 88:10535; Zheng et al., 1995, J Immunol154:5590; and Vil et al., 1992, PNAS USA 89:11337; each incorporated byreference in their entireties. The fusion of an antibody, or fragmentthereof, to a moiety does not necessarily need to be direct, but mayoccur through linker sequences. Such linker molecules are commonly knownin the art and described in Denardo et al., 1998, clin Cancer Res4:2483; Peterson et al., 1999, Bioconjug Chem 10:553; Zimmerman et al.,1999, Nucl Med Biol 26:943; Garnett, 2002, Adv Drug Deliv Rev 53:171.

In one embodiment, antibodies, or fragments thereof, isolated inaccordance with the present invention are recombinantly fused orchemically conjugated (including both covalent and non-covalentconjugations) to a heterologous protein or polypeptide (or fragmentthereof, preferably to a polypeptide of at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90 or at least 100 amino acids) to generate fusionproteins. Alternatively, or optionally, antibodies, or fragmentsthereof, may be used to target heterologous polypeptides to particularcell types, either in vitro or in vivo, by fusing or conjugating theantibodies to antibodies specific for particular cell surface receptors.Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

7.9 Transmembrane Domains

In the present invention, a coding sequence for a transmembrane domainis located downstream of and operatively linked to the first codingregion (FIG. 1). Therefore, a fusion protein is expressed that iscapable of localizing onto the cell membrane where it can be detectedusing techniques well known in the art.

Transmembrane regions of proteins are highly hydrophobic or lipophilicdomains that are the proper size to span the lipid bilayer of thecellular membrane, thereby anchoring proteins, peptides, or receptors inthe cell membrane. They will typically, but not always, comprise 15-30amino acids. See Chou et al. (1999 Biotechnology and Bioengineering65(2):160-169), which describes using several transmembrane domains fromdifferent source proteins to express a different protein on the cellmembrane. One skilled in the art can adapt the method performed in Chouet al. to optimize or screen different transmembrane domains and/orGPI-anchor domains for use in the present invention.

Transmembrane proteins may contain from one or multiple transmembranedomains. For example, receptor tyrosine kinases, certain cytokinereceptors, receptor guanylyl cyclases and receptor serine/threonineprotein kinases contain a single transmembrane domain. However, variousother proteins including channels and adenylyl cyclases contain numeroustransmembrane domains.

Many cell surface receptors are classified as “seven transmembranedomain” proteins, as they contain membrane spanning regions.Transmembrane protein receptors include, but are not limited to insulinreceptor, insulin-like growth factor receptor, human growth hormonereceptor, glucose transporters, transferrin receptor, epidermal growthfactor receptor, low density lipoprotein receptor, epidermal growthfactor receptor, leptin receptor, interleukin receptors, e.g. IL-1receptor, IL-2 receptor, etc.

Various approaches in eukaryotic systems achieve surface display byproducing fusion proteins that contain the polypeptide of interest and atransmembrane domain from another protein to anchor the fusion proteinto the cell membrane. Not wishing to be bound by theory, in eukaryoticcells, the majority of secreted proteins and membrane-bound proteins aretranslocated across an endoplasmic reticulum membrane concurrently withtranslation (Wicker and Lodish, Science 230:400 (1985); Verner andSchatz, Science 241:1307 (1988); Hartmann et al., Proc. Nat'l Acad. Sci.USA 86:5786 (1989); Matlack et al., Cell 92:381 (1998)). In the firststep of this co-translocational process, an N-terminal hydrophobicsegment of the nascent polypeptide, called the “signal sequence,” isrecognized by a signal recognition particle and targeted to theendoplasmic reticulum membrane by an interaction between the signalrecognition particle and a membrane receptor. The signal sequence entersthe endoplasmic reticulum membrane and the following nascent polypeptidechain begins to pass through the translocation apparatus in theendoplasmic reticulum membrane. The signal sequence of a secretedprotein or a type I membrane protein is cleaved by a signal peptidase onthe luminal side of the endoplasmic reticulum membrane and is excisedfrom the translocating chain. The rest of the secreted protein chain isreleased into the lumen of the endoplasmic reticulum. A type I membraneprotein is anchored in the membrane by a second hydrophobic segment,which is usually referred to as a “transmembrane domain.” The C-terminusof a type I membrane protein is located in the cytosol of the cell,while the N-terminus is displayed on the cell surface.

As used herein, the term “type II signal anchor domain,” or “type IItransmembrane domain,” refers to a hydrophobic amino acid sequence foundin eukaryotic type II integral membrane proteins that, duringtranslation, targets and anchors a polypeptide in the endoplasmicreticulum membrane with a type II orientation. The phrase “type IIorientation,” refers to a protein topology in which the N-terminusresides in the cytoplasm, while the C-terminus resides within the lumenof the endoplasmic reticulum or on an extracellular cell surface.

In contrast, certain proteins have a signal sequence that is notcleaved, a “signal anchor sequence,” which serves as a transmembranesegment. A signal anchor type I protein has a C-terminus that is locatedin the cytosol, which is similar to type I membrane proteins, whereas asignal anchor type II protein has an N-terminus that is located in thecytosol. Examples of type 11 signal anchors are described in, forexample, Yokoyarna-Kobayashi et al., Gene 228:161 (1999).

In one embodiment, the transmembrane domain is from a type I membraneprotein.

Described herein are examples of transmembrane domains, but thetransmembrane domain of the fusion proteins of the invention can be anyamino acid sequence that will span the plasma cell membrane and cananchor other domains to the membrane. Characteristics of transmembranedomains include generally consecutive hydrophobic amino acids that maybe followed by charged amino acids. Therefore, upon analysis of theamino acid sequence of a particular protein, the localization and numberof transmembrane domains within the protein may be predicted by thoseskilled in art. A transmembrane domain may comprise hydrophobic regionsor amphipathic regions. Hydrophobic regions contain hydrophobic aminoacids, which include, but are not limited to, phenylalanine, methionine,isoleucine, leucine, valine, cysteine, tryptophan, alanine, threonine,glycine and serine and include hydrophobic alpha-helices.

Amphipathic regions may have both hydrophobic and hydrophilic aminoacids and moieties and include amphipathic alpha-helices. Hydrophilicamino acids include, but are not limited to, arginine, aspartate,lysine, glutamate, asparagine, glutamine, histidine, tyrosine andproline. Transmembrane domains that form stable alpha helices have beenpreviously described in the art.

Essentially any transmembrane domain is compatible with the presentinvention. Transmembrane domains include, but are not limited to, thosefrom: a member of the tumor necrosis factor receptor superfamily, CD30,platelet derived growth factor receptor (PDGFR, e.g. amino acids 514-562of human PDGFR; Chestnut et al. 1996 J Immunological Methods 193:17-27;also see Gronwald et al. 1988 PNAS 85:3435); nerve growth factorreceptor, Murine B7-1 (Freeman et al. 1991 J Exp Med 174:625-631),asialoglycoprotein receptor H1 subunit (ASGPR; Speiss et al. 1985 J BiolChem 260:1979-1982), CD27, CD40, CD120a, CD120b, CD80 (Freeman et al.1989 J Immunol 143:2714-22) lymphotoxin beta receptor,galactosyltransferase (E.G. GenBank accession number AF155582), sialylytransferase (E.G. GenBank accession number NM-003032), aspartyltransferase 1 (Asp1; e.g. GenBank accession number AF200342), aspartyltransferase 2 (Asp2; e.g. GenBank accession number NM-012104), syntaxin6 (e.g. GenBank accession number NM-005819), ubiquitin, dopaminereceptor, insulin B chain, acetylglucosaminyl transferase (e.g. GenBankaccession number NM-002406), APP (e.g. GenBank accession number A33292),a G-protein coupled receptor, thrombomodulin (Suzuki et al. 1987 EMBO J6, 1891) and TRAIL receptor. In one embodiment, the transmembrane domainis from a human protein. For the purposes of the present invention allor part of a transmembrane domain from a proteins may be utilized. Inspecific embodiments, the transmembrane domain is residues 454-477 ofthe Asp2, residues 598-661 of APP (e.g., of APP 695), residues 4-27 ofgalactosyltransferase, residues 470-492 of Asp1, residues 10-33 ofsialyltransferase, residues 7-29 of acetylglucosaminyl transferase orresidues 261-298 of syntaxin 6. Examples of transmembrane domains arealso described in Patent Publications WO 03/104415 and US20040126859. Inone embodiment, the transmembrane domain is derived from a humanprotein, e.g., described herein.

In one embodiment, a cell surface displayed antibody or fragment thereofof the current invention comprises the transmembrane domain ofthrombomodulin having an amino acid sequence ofLLIGISIASLCLVVALLALLCHLRKKQ (SEQ ID NO:109).

In one embodiment, the pDisplay™ vector from Invitrogen (Carlsbad,Calif.; Catalog no. V660-20) is used during one of the cloning steps forconstructing the viral vector. The pDisplay™ vector is a mammalianexpression vector designed to target recombinant proteins to the surfaceof mammalian cells. Proteins of interest are targeted and anchored tothe cell surface by cloning the gene of interest in frame with thevector's N-terminal secretion signal and the C-terminal transmembraneanchoring domain of platelet-derived growth factor receptor (PDGFR). Forfurther details see the product manual titled “pDisplay ™ Vector forexpression of proteins on the surface of mammalian cells” Version C fromInvitrogen.

7.10 GPI-anchor Signal Sequence

A wide range of cell-surface proteins, including enzymes, coat proteins,surface antigens, and adhesion molecules, are attached to plasmamembranes via GPI anchors (Burikofer et al. 2002 FASEB J 15:545). GPI isa post-translationally added lipid anchor; therefore, unlikeconventional polypeptide anchors which have different transmembranedomains and connect to specific cytoplasmic extensions, GPI anchors usea common lipid structure to attach to the membrane, which isirrespective of the proteins linked with it (Englund et al., Annul Rev.Biochem. 62:121 (1993)). GPI anchor signal sequences have beenidentified for many proteins (for example, see Cares et al., Science243:1196 (1989)). The GPI anchor signals have been successfullyengineered onto the C-terminus of other un-GPI anchored proteins, andthese GPI anchored proteins are coated on the cell surface and arefunctional. (Anderson et al., P.N.A.S. 93:5894 (1996); Brunschwig etal., J. Immunother. 22:390 (1999)). GPI anchors are proposed to functionin protein targeting, transmembrane signaling, and in the uptake ofsmall molecules (endocytosis). GPI anchors of plasma membrane proteinsare present in eukaryotes from protozoa and fungi to vertebrates. Forexamples of GPI anchor domains, which may be utilized in the presentinvention, see Doering, T. L. et al. (1990) J. Biol. Chem. 265:61 1-614;McConville, M. J. et al. (1993) Biochem. J. 294:305-324; and PCTPublication WO 03/017944).

Without wishing to be limited by theoretical considerations, immediatelyfollowing protein synthesis, a protein comprising a GPI modificationsignal is anchored to the ER lumen by a hydrophobic sequenceapproximately 15-20 amino acids in length. Alberts et al., MolecularBiology Of The Cell, 3rd Edition, p. 591 (1994). A GPI anchor ispre-assembled in the ER and following GPI attachment, the modifiedprotein is glycosylated and shuttled to the exterior surface of theplasma membrane. The process of covalently attaching a GPI anchor to theC-terminus of a peptide is catalyzed by enzymes in the rough ER. Enzymesof the ER cleave the original membrane-anchor sequence and then the newcarboxyl-terminus is attached to the amino group of ethanolamine. Theanchor typically comprises a phosphoethanolamine (EthN-P), severalsugars, including N-acetylglucosamine (GlcNAc) and mannose, linked to aninositol phospholipid (Ikezawa 2002 Biol Pharm Bull 25: 409-417).Furthermore, the inositol phospholipid typically contains 1-alkyl,2-acyl glycerol. The inositol phospholipids in anchors, however, canvary. For example, inositol phospholipids of proteins expressed onerythrocytes have an additional inositol-associated fatty acid thatprovides an additional point of attachment to the plasma membrane. Suchanchors are described as being “two footed.” Accordingly, the GPIanchors of to the present invention can be “one footed,” “two footed” or“three footed”.

There are some general requirements for creating a synthetic GPI anchorsequence. These are a hydrophobic region at the C-terminus of themolecule (10-20 amino acids) not followed by a cluster of basicresidues, a “spacer domain” of 7-10 residues preceding the hydrophobicregion and small amino acids after the spacer region, where cleavage ofthe precursor and attachment of the anchor occurs. The GPI anchor ispreassembled and added to nascent protein in the endoplasmic reticulum(ER). Concomitant with this step, the initial C-terminal peptide isremoved so that the GPI anchor is covalently attached to a newC-terminal amino acid on the protein.

The present invention utilizes a GPI-anchor signal sequence to expressan antibody on a cell membrane as described herein. The GPI anchorsignal sequence coding region is located downstream of and operativelylinked to the first coding region. Therefore, a fusion protein isexpressed that is capable of localizing onto the cell membrane where itcan be detected using techniques well known in the art (FIG. 1).

It is thought that GPI-anchored proteins also utilize a N-terminalsignal sequence that directs the protein to the ER. This signal can beengineered into the coding region by common methods known in the art.

Essentially any GPI-anchor signal sequence can be used in accordancewith the invention. GPI-anchor signal sequences are known in the artand/or can be determined using methods known in the art, e.g. using theBig-P predictor analys is available through the official website of theIMP Bioinformatics Group and described in Eisenhaber et al. 1999 J MolBiol 292: 741-758; Eisenhaber et al. 2003 Nucleic Acids Research 31:3631-3634; Eisenhaber et al. Protein Engineering 14: 17-25; Eisenhaberet al. 2000 TIBS 25: 340-341; and Eisenhaber et al. 1998 ProteinEngineering 11: 1155-1161.

In one embodiment, the GPI-anchor signal sequence is selected from aGPI-anchor signal from a eukaryotic, mammalian, primate or humanprotein. GPI-anchor signal sequences include, but are not limited to,those from decay accelerating factor (DAF; Caras et al. 1987 Science238:1280-83); uromodulin, alkaline phosphatase, BP-3,dipeptidylpeptidase, Trypanosoma brucei variant surface protein (VSG;Doering et al. J Bio Chem. 1990 256:611-614); C8 binding protein(Doering et al. 1990); Alkaline phosphatase; Acetylcholinesterase;59-Nucleotidase; Alkalinephosphodiesterase I; Trehalase; Leishmaniasurface protease PSP (gp63); Renal dipeptidase (MDP); Aminopeptidase P;NAD 1 glycohydrolase; Carboxypeptidase M; Carbonic anhydrase IV;Silkworm aminopeptidaseN; ADP-ribosyltransferase; Yeast aspartylprotease; Chlorella nitrate reductase; Plasmodium transferrin receptor;CD14; CD16; CD48; Folate-binding protein; Urokinase receptor; CNTFreceptor; Trypanosoma VSG and PARP (procyclin); Toxoplasma surfaceantigens(P22; P30 and P43); Giardia GP49; Paramecium surface antigens;Thy-1; CD55 (DAF); Ly6 family (CD59; Ly6A/E); Carcinoembryonic antigen(CEA); Qa-2; CD24, Prions (PrP C ; PrP Sc); Squid Sgp-1 and Sgp-2;NCAM-120 (the shortest CD56); CD58 (LFA-3; Seed et al. 1987 Nature329:840-842); Dictyostelium Contact site A; Mouse F3; ChickF11; Chickenaxonin-1; Polysphondylium GP64; Grasshopper REGA-1; 5NTD_BOVIN;5NTD_DISOM; 5NTD_HUMAN; 5NTD_RAT; ACES_TORCA; ACES_TORMA; AMPM_HELVI;AMPM_MANSE; AXO1_HUMAN; BCM1_HUMAN; BCM1_MOUSE; BCM1_RAT; BM86_BOOMI;BST1_HUMAN; BST1_MOUSE; BST1_RAT; CADD_CHICK; CADD_HUMAN; CAH4_HUMAN;CD24_HUMAN; CD24_MOUSE; CD24_RAT; CD48_HUMAN; CD48_MOUSE; CD48_RAT;CD52_HUMAN; CD52_MACFA; CD59_AOTTR; CD59_CALSQ; CD59_CERAE; CD59_HSVSA;CD59_HUMAN; CD59_PAPSP; CD59_PIG; CD59_RAT; CD59_SAISC; CEPU_CHICK;CGM6_HUMAN; CNTR_CHICK; CNTR_HUMAN; CNTR_RAT; CONN_DROME; CSA_DICDI;CWP1_YEAST; CWP2_YEAST; DAF_HUMAN; DAF_PONPY; DAF1_MOUSE; FOL1_HUMAN;FOL1_MOUSE; FOL2_HUMAN; FOL2_MOUSE; G13A_DICDI; G13B_DICDI; GAS1_YEAST;GLYP_HUMAN; GLYP_RAT; GP46_LEIAM; GP63_LEICH; GP63_LEIDO; GP63_LEIGU;GP63_LEIMA; GP85 TRYCR; GPCK_MOUSE; HYA1_CAVPO; HYA1_HUMAN; HYA1_MACFA;HYR1_CANAL; LACH_DROME; LACH_SCHAM; LAMP_HUMAN; LAMP_RAT; LY6A_MOUSE;LY6C_MOUSE; LY6E_MOUSE; LY6F_MOUSE; LY6G_MOUSE; MDP1_HUMAN; MDP1_MOUSE;MDP1_PIG; MDP1_RABIT; MDP1_RAT; MDP1_SHEEP; MKC7_YEAST; MSA1_SARMU;NAR3_HUMAN; NART_MOUSE; NCA_HUMAN; NRT1_RAT; NRT2_RAT; NRTR_CHICK;NRTR_HUMAN; NRTR_MOUSE; NTR1_RAT; OPCM_BOVIN; OPCM_HUMAN; OPCM_RAT;PAG1_TRYBB; PARA_TRYBB; PARB_TRYBB; PARC_TRYBB; PONA_DICDI; PPB1_HUMAN;PPB2_HUMAN; PPB3_HUMAN; PPBE_MOUSE; PPBI_BOVIN; PPBI_HUMAN; PPBI_RAT;PPBJ_RAT; PRIO_ATEGE; PRIO_ATEPA; PRIO_CALJA; PRIO_CEBAP; PRIO_CERAE;PRIO_CERAT; PRIO_CERMO; PRIO_CERNE; PRIO_CERPA; PRIO_CERTO; PRIO_COLGU;PRIO_CRIGR; PRIO_CRIMI; PRIO_GORGO; PRIO_HUMAN; PRIO_MACFA; PRIO_MACSY;PRIO_MANSP; PRIO_MESAU; PRIO_MOUSE; PRIO_PANTR; PRIO_PONPY; PRIO_PREFR;PRIO_RAT; PSA_DICDI; SP63_STRPU; THY1_CHICK; THY1_HUMAN; THY1_MACMU;THY1_MOUSE; THY1_RAT; TIP1_YEAST; TIR1_YEAST; TREA_HUMAN; TREA_RABIT;UPAR_BOVIN; UPAR_HUMAN; UPAR_MOUSE; UPAR_RAT; VCA1_MOUSE; VSA1_TRYBB;VSA8 TRYBB; VSAC_TRYBB; VSE2_TRYBR; VSG2_TRYEQ; VSG4_TRYBR; VSG7_TRYBR;VSI1_TRYBB; VSI2_TRYBB; VSI3_TRYBB; VSI4_TRYBB; VSI5_TRYBB; VSI6_TRYBB;VSIB_TRYBB; VSM0_TRYBB; VSM1_TRYBB; VSM2_TRYBB; VSM4_TRYBB; VSM5_TRYBB;VSM5_TRYBR; VSM6_TRYBB; VSWA_TRYBR; VSWB_TRYBR; VSY1_TRYCO; YAP3_YEAST;YJ9O_YEAST; and YJ9P_YEAST. In one embodiment, the GPI-anchor signalsequence is the C-terminal 37 amino acids of DAF. For further examplesof GPI-anchored signal sequences see U.S. Pat. No. 5,968,742 and Doeringet al. 1990, supra. Examples of specific GPI-anchored signal include,but are not limited to, those listed in Table 2.

TABLE 2 Examples of Signal Peptides for GPI-anchoring. SEQ ID Amino AcidSequence NO DKLVKCGGISLLVQNTSWMLLLLLSLSLLQALDFISL 56PSPTPTETATPSPTPKPTSTPEETEAPSSATTLISPLSLIVIFISF 57 VLLILVPRGSIEGRGTSITAYNSEGESAEFFFLLILLLLLVLV 58 TSITAYKSEGESAEFFFLLILLLLLVLV59 SNKGSGTTSGTARLLSGHTCFTLTGLLGTLVIMGLLT 60PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT 61 PDHSAATKPSLFLFLVSLLHIFFK 627.11 Internal Ribosome Entry Sites

IRESs are used to express two or more proteins from a single vector. AnIRES sequence is commonly used to drive expression of a second, third,fourth coding sequence, etc.

IRES elements were first discovered in picornavirus mRNAs (Jackson etal., 1990, Trends Biochem Sci 15:477-S3; Jackson et al., 1995, RNA1:985-1000). Examples of IRESs that can be used in accordance with thepresent invention include, but are not limited to, those from or derivedfrom Picornavirus e.g., HAV (Glass et al. 1993, Virol 193:842-852),encephelomycarditis virus (EMCV) which is e.g., commercially availablefrom Novagen (Duke et al., 1992, J. Virol 66:1602-9; Jang & Wimmer,1990, Gene Dev 4:1560-1572), and Poliovirus (Borman et al., 1994, EMBO J13:3149-3157); HCV (Tsukiyama-Kohara et al., 1992, J Virol 66:1476-1483)BVDV (Frolov I et al., 1998, RNA. 4:1418-1435); Leishmania virus, e.g.,LRV-1 (Maga et al., 1995, Mol Cell Biol 15:4884-4889); Retrovirusese.g., MoMLV (Torrent et al., 1996, Hum Gene Ther 7:603-612), VL30(Harvey murine sarcoma virus), REV (Lopez-Lastra et al., 1997, Hum GeneTher 8:1855-1865); and Eukaryotic mRNA e.g. immunoglobulin heavy-chainbinding protein (BiP) (Macejak & Sarnow, 1991, Nature 353:90-94),antennapedia mRNA (Oh et al., 1992, Gene & Dev 6:1643-1653), fibroblastgrowth factor 2 (FGF-2) (Vagner et al., 1995, Mol Cell Biol 15:35-44),PDGF-B (Bernstein et al., 1997, J Biol Chem 272:9356-9362), IGFII(Teerink et al., 1995, Biochim Biophys Acta 1264:403-408), translationalinitiation factor eIF4G (Gan & Rhoads, 1996, J Biol Chem 271:623-626),insulin-like growth factor (IGFU), yeast transcription factors TFIID andHAP4, and the vascular endothelial growth factor (VEGF) (Stein et al.,1998, Mol Cell Biol 18:3112-3119; Huez et al., 1998, Mol Cell Biol18:6178-6190) as well as those described in U.S. Pat. No. 6,692,736.IRESs have also been reported in different viruses such as cardiovirus,rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV) andMoloney murine I leukemia virus (MoMLV). As used herein, the term “IRES”encompasses functional variations of IRES sequences as long as thevariation is able to promote direct internal ribosome entry to theinitiation codon of a downstream cistron, leading to cap-independenttranslation. An IRES utilized in the present invention may be mammalian,viral or protozoan.

Thus, the product of a downstream cistron can be expressed from abicistronic (or multicistronic) mRNA, without requiring either cleavageof a polyprotein or generation of a monocistronic mRNA. Commonly usedinternal ribosome entry sites are approximately 450 nucleotides inlength and are characterized by moderate conservation of primarysequence and strong conservation of secondary structure. The mostsignificant primary sequence feature of the IRES is a pyrimidine-richsite, whose start is located approximately 25 nucleotides upstream ofthe 3′ end of the IRES. See Jackson et al., 1990 (Trends Biochem Sci,15(12):477-83).

Three major classes of picomavirus IRES have been identified andcharacterized: (1) the cardio- and aphthovirus class (for example, theencephelomycarditis virus, Jang et al., 1990, Gene Dev 4:1560-1572); (2)the entero- and rhinovirus class (for example, polioviruses, Borman etal., 1994, EMBO J. 13:314903157); and (3) the hepatitis A virus (HAY)class, Glass et al., 1993, Virol 193:842-852). For the first twoclasses, two general principles apply. First, most of the about450-nucleotide sequence of the IRES functions to maintain particularsecondary and tertiary structures conducive to ribosome binding andtranslational initiation. Second, the ribosome entry site is an AUGtriplet located at the 3′ end of the IRES, approximately 25 nucleotidesdownstream of a conserved oligopyrimidine tract. Translation initiationcan occur either at the ribosome entry site (cardioviruses) or at thenext downstream AUG (entero/rhinovirus class). Initiation occurs at bothsites in aphthoviruses.

HCV and pestiviruses such as bovine viral diarrhea virus (BVDV) or;classical swine fever virus (CSFV) have 341 nt and 370 nt long 5′-UTRrespectively. These 5′-UTR fragments form similar RNA secondarystructures and can have moderately efficient IRES function(Tsukiyama-Kohara et al., 1992, J. Virol. 66:1476-1483; Frolov I et al.,1998, RNA 4:1418-1435). Recent studies showed that both Friend-murineleukemia virus (MLV) 5′-UTR and rat retrotransposon virus-like 30S(VL30) sequences contain IRES structure of retroviral origin (Torrent etal., 1996, Hum Gene Ther 7:603-612).

In eukaryotic cells, translation is normally initiated by the ribosomescanning from the capped mRNA 5′ end, under the control of initiationfactors. However, several cellular mRNAs have been found to have IRESstructure to mediate the cap-independent translation (van der Velde, etal., 1999, Int J Biochem Cell Biol. 31:87-106). Non-limiting examplesare: immunoglobulin heavy-chain binding protein (BiP) (Macejak et al.,1991, Nature 353:90-94), antennapedia mRNA of Drosophila (Oh et al.,1992, Gene & Dev 6:1643-1653), fibroblast growth factor 2 (FGF-2)(Vagner et al., 1995, Mol Cell Biol 15:35-44), platelet-derived growthfactor B (PDGF-B) (Bernstein et al., 1997, J Biol Chem 272:9356-9362),insulin-like growth factor II (Teerink et al., 1995, Biochim BiophysActa 1264:403-408), the translation initiation factor eIF4G (Gan &Rhoads, 1996, J Biol Chem 271:623-626) and vascular endothelial growthfactor (VEGF) (Stein et al., 1998, Mol Cell Biol 18:3112-3119; Huez etal., 1998, Mol Cell Biol 18:6178-6190) .

An IRES may be prepared using standard recombinant and synthetic methodsknown in the art. For cloning convenience, restriction sites may beengineered into the ends of the IRES fragments to be used.

7.12 Self-Processing cleavage Sites Or Sequences

Although the mechanism is not part of the invention, the activity ofself-processing cleavage site, self-processing cleavage sequence or a2A-like sequence may involve ribosomal skipping between codons whichprevents formation of peptide bonds (de Felipe et al., 2000, Human GeneTherapy 11: 1921-1931; Donnelly et al., 2001, J. Gen. Virol.82:1013-1025), although it has been considered that the domain acts morelike an autolytic enzyme (Ryan et al., 1989, Virol. 173.35-45).

A “self-processing cleavage site” or “self-processing cleavage sequence”refers to a DNA or amino acid sequence, wherein upon translation, rapidintramolecular (cis) cleavage of a polypeptide comprising theself-processing cleavage site occurs to result in expression of discretemature protein or polypeptide products. Also, a “self-processingcleavage site” or “self-processing cleavage sequence” refers to a DNA oramino acid sequence, wherein upon translation, the sequence results in“ribosomal skip” as known in the art and described herein. A“self-processing cleavage site”, may also be referred to as apost-translational or co-translational processing cleavage site,exemplified herein by a 2A site, sequence or domain. It has beenreported that a 2A site, sequence or domain demonstrates a translationaleffect by modifying the activity of the ribosome to promote hydrolysisof an ester linkage, thereby releasing the polypeptide from thetranslational complex in a manner that allows the synthesis of adiscrete downstream translation product to proceed (Donnelly et al.,2001, J Gen Virol. 82:1013-25). Alternatively, a “self-processingcleavage site”, “self-processing cleavage sequence” or a 2A sequence ordomain demonstrates “auto-proteolysis” or “cleavage” by cleaving its ownC-terminus in cis to produce primary cleavage products (Furler;Palmenberg, 1990, Ann. Rev. Microbiol. 44:603-623).

Although the mechanism is not part of the invention, the activity of a2A-like sequence or self-processing cleavage site may involve ribosomalskipping between codons which prevents formation of peptide bonds (deFelipe et al., 2000, Human Gene Therapy 11: 1921-1931; Donnelly et al.,2001, J. Gen. Virol. 82:1013-1025), although it has also been consideredthat the domain acts more like an autolytic enzyme (Ryan et al., Virol.173.35-45 (1989).

The Foot and Mouth Disease Virus 2A oligopeptide has previously beendemonstrated to mediate the translation of two sequential proteinsthrough a ribosomal skip mechanism (Donnelly et al., 2001, J Gen Virol.82:1013-25; Szymczak et al., 2004, Nat Biotechnol. 5:589-94.; Klump etal., 2001, Gene Ther. 10:811-7; De Felipe et al., 2000, Hum Gene Ther.11:1921-31; Halpin et al., 1999, Plant J. 17:453-9; Mattion et al.,1996, J Virol. 70:8124-7; and de Felipe P. et al., 1999, Gene Ther.6:198-208). Multiple proteins are encoded as a single open reading frame(ORF). During translation in a bicistronic system, the presence of theFMDV 2A sequence at the 3′ end of the upstream gene abrogates thepeptide bond formation with the downstream cistron, resulting in a“ribosomal skip” and the attachment of the translated FMDV 2Aoligopeptide to the upstream protein (Donnelly et al., 2001, J GenVirol. 82(Pt 5): 1013-25). Processing occurs in a stoichiometricfashion, estimated to be as high as 90-99%, resulting in a near molarequivalency of both protein species (Donnelly et al., 2001, J Gen Virol.82(Pt 5):1027-41). Furthermore, through deletion analysis the amino acidsequence-dependent processing activity has been localized to a smallsection at the c-terminal end of the FMDV 2A oligopeptide (Ryan et al.,1994, EMBO J. 13:928-33). Most members of the Picornavirus family (ofwhich FMDV belongs) use similar mechanisms of cotranslational processingto generate individual proteins (Donnelly et al., 2001, J Gen Virol.82(Pt 5):1027-41). In fact, publications have shown that fragments assmall as 13 amino acids can cause the ribosomal skip (Ryan et al., 1994,EMBO J. 13:928-33). Incorporation of truncated versions of the peptidein bicistronic vector systems has demonstrated that almost all of theprocessing activity is preserved even in non-viral vector systems(Donnelly et al., 2001, J Gen Virol. 82(Pt 5):1027-41). At least fourcoding sequences that have been efficiently expressed under a singlepromoter by strategic placement of these types of elements (Szymczak etal., 2004, Nat Biotechnol. 22:589-94.). Therefore, self-processingcleavage sites such as the FMDV 2A oligopeptide may be utilized in thepresent invention to link expression of the heavy and light chain codingregions.

For the present invention, the DNA sequence encoding a self-processingcleavage site is exemplified by viral sequences derived from apicornavirus, including but not limited to an entero-, rhino-, cardio-,aphtho- or Foot-and-Mouth Disease Virus (FMDV). In one embodiment, theself-processing cleavage site coding sequence is derived from a FMDV.

The FMDV 2A domain is typically reported to be about nineteen aminoacids in length (e.g., LLNFDLLKLAGDVESNPGP (SEQ ID NO: 56);TLNFDLLKLAGDVESNPGP (SEQ ID NO: 57), Ryan et al., J. Gen. Virol.72.2727-2732 (1991)), however oligopeptides of as few as thirteen aminoacid residues (e.g., LKLAGDVESNPGP (SEQ ID NO: 58)) have also been shownto mediate cleavage at the 2A C-terminus in a fashion similar to itsrole in the native FMDV polyprotein processing. Alternatively, a vectoraccording to the invention may encode amino acid residues for other2A-like regions as discussed in Donnelly et al., 2001, J. Gen. Virol.82:1027-1041 and including but not limited to a 2A-like domain frompicomavirus, insect virus, Type C rotavirus, trypanosome repeatedsequences or the bacterium, Thermatoga maritime.

Variations of the 2A sequence have been studied for their ability tomediate efficient processing of polyproteins (Donnelly et al., 2001) .Such variants are specifically contemplated and encompassed by thepresent invention. In one embodiment, the 2A sequence is a variant 2Asequence.

Further examples and descriptions of self-processing cleavage sites andvectors encoding them are found in US20050042721 and US2005003482.

7.13 Specific Embodiments

Additional embodiments of the present invention are presented in Table3.

TABLE 3 Specific embodiments. 1 A recombinant antibody or fragmentthereof that is displayed on the extracellular surface of the cellmembrane. 2 The antibody or fragment thereof of embodiment 1, comprisingan amino acid sequence that targets the antibody to the cell surfacewherein said amino acid sequence is fused to the heavy chain or thelight chain of the antibody. 3 The antibody or fragment thereof ofembodiment 2, wherein said amino acid sequence is fused to theC-terminal end of the heavy chain or the light chain of the antibody. 4The antibody or fragment thereof of embodiment 2, wherein said aminoacid sequence comprises a transmembrane domain or a GPI anchor signalsequence. 5 The antibody or fragment thereof of embodiment 4, whereinsaid transmembrane domain is derived from thrombomodulin. 6 The antibodyor fragment thereof of embodiment 5, wherein said transmembrane domaincomprises SEQ ID NO: 109. 7 The antibody or fragment thereof ofembodiment 4, wherein said GPI anchor domain is derived from DAF. 8 Theantibody or fragment thereof of embodiment 7, wherein said GPI anchordomain comprises SEQ ID NO: 60 or 61. 9 The antibody or fragment thereofof embodiment 1, wherein said antibody or fragment thereof is from animmunoglobulin type selected from the group consisting of IgA, IgE, IgM,IgD, IgY and IgG. 10 The antibody or fragment thereof of embodiment 1,wherein said antibody or fragment thereof is a murine antibody, achimeric antibody, a humanized antibody or human antibody. 11 Theantibody or fragment thereof of embodiment 1, wherein said antibody orfragment thereof is a human antibody. 12 The antibody or fragmentthereof of embodiment 1, wherein said antibody or fragment thereofcomprises an Fc region. 13 The antibody or fragment thereof ofembodiment 1 wherein said antibody or fragment thereof comprises a heavychain variable region, a light chain variable region or both a heavychain and a light chain variable region. 14 A polynucleotide encodingthe antibody or fragment thereof of any one of embodiments 1-13. 15 Avector comprising the polynucleotide sequence of embodiment 14. 16 Thevector of embodiment 15, further comprising a polyadenylation signalsequence. 17 The vector of embodiment 16, wherein said polyadenylationsignal sequence is selected from the group consisting of bovine growthhormone polyA signal sequence and SV40 polyA signal sequence. 18 Thevector of embodiment 15, further comprising a promoter. 19 The vector ofembodiment 18, wherein said promoter is a CMV or RSV promoter. 20 Thevector of embodiment 15, further comprising an IRES or self processingcleavage site. 21 The vector of embodiment 15, wherein said vector iscapable of replication. 22 The vector of embodiment 21, wherein saidvector is a viral vector. 23 The vector of embodiment 22, wherein saidviral vector is an adenoviral vector, a baculoviral vector, an adenoassociated viral vector, a herpes viral vector or a lentiviral vector.24 The vector of embodiment 22, wherein said vector is an adenoviralvector. 25 A cell comprising the vector of embodiment 14. 26 The cell ofembodiment 25, wherein said cell is a mammalian cell. 27 The cell ofembodiment 26, wherein said cell is selected from the group consistingof a NS0 cell, a CHO cell, a Vero cell, an Sf-9 cell, a COS7 cell, and a293 cell. 28 The cell of embodiment 25, wherein said cell is a humancell. 29 A library of vectors comprising polynucleotides encodingrecombinant antibodies or fragments thereof that are displayed on theextracellular surface of the cell membrane. 30 The library of embodiment29, wherein said antibodies or fragments thereof comprise Fc regionvariants. 31 The library of embodiment 29, wherein said antibodies orfragments thereof comprise a library of light chain variable regionsequences. 32 The library of embodiment 29, wherein said antibodies orfragments thereof comprise a library of heavy chain variable regionsequences. 33 The library of embodiment 29, wherein said antibodies orfragments thereof comprise a library of light chain variable regionsequences and a library of heavy chain variable region sequences. 34 Thelibrary of embodiment 29, 30, 31, 32 or 33, wherein said antibodies orfragments thereof comprise an amino acid sequence that targets theantibody to the cell surface wherein said amino acid sequence is fusedto the heavy chain or the light chain of the antibody. 35 The library ofembodiment 34, wherein said amino acid sequence is fused to theC-terminal end of the heavy chain or the light chain of the antibody. 36The library of embodiment 34, wherein said amino acid sequence comprisesa transmembrane domain or a GPI anchor signal sequence. 37 The libraryof embodiment 36, wherein said transmembrane domain is derived fromthrombomodulin. 38 The library of embodiment 37, wherein saidtransmembrane domain comprises SEQ ID NO: 109. 39 The library ofembodiment 36, wherein said GPI anchor domain is derived from DAF. 40The library of embodiment 39, wherein said GPI anchor domain comprisesSEQ ID NO: 60 or 61. 41 The library of embodiment 34, wherein saidvectors are capable of replication. 42 The library of embodiment 41,wherein said vectors are viral vectors. 43 The library of embodiment 41,wherein said viral vectors are adenoviral vectors, baculoviral vectors,adeno associated viral vectors, herpes viral vectors or lentiviralvectors. 44 The library of embodiment 42, wherein said vectors areadenoviral vectors. 45 A population of cells comprising the library ofembodiment 34. 46 The cells of embodiment 45, wherein said cells aremammalian cells. 47 The cells of embodiment 46, wherein said cells areselected from the group consisting of NS0 cells, CHO cells, Vero cells,Sf-9 cells, COS7 cells, and 293 cells. 48 A method of isolating anantibody or fragment thereof having a desirable characteristiccomprising: a) culturing the population of cells of embodiment 45 underconditions that allow expression of the antibodies on the cell surface;b) subjecting the population of cells to selection thereby isolating atleast one cell expressing an antibody or fragment thereof having thedesired characteristic. 49 The method of embodiment 48, furthercomprising the step of isolating a polynucleotide from the selected cellwherein said polynucleotide encodes the antibody or fragment thereofhaving a desirable characteristic. 50 The method of embodiment 48,wherein said desirable characteristic is binding to a specific antigen.51 The method of embodiment 48, wherein said desirable characteristic isincreased binding to a specific antigen. 52 The method of embodiment 48,wherein said desirable characteristic is decreased binding to a specificantigen. 53 The method of embodiment 48, wherein said desirablecharacteristic is binding to an effector molecule. 54 The method ofembodiment 48, wherein said desirable characteristic is reduced bindingto an effector molecule. 55 The method of embodiment 48, wherein saiddesirable characteristic is increased binding to an effector molecule.56 The method of any one of embodiments 53 to 55, wherein said effectormolecule is selected from the group consisting of C1q, FcγRI, FcγRII andFcγRIIIA. 57 The method of embodiment 48, wherein the selection iscarried out by incubating the cells with a labeled reagent and sortingthe cells based on the binding of the reagent to the cells. 58 A methodfor producing a library of cells displaying antibodies or antibodyfragments on the cell surface comprising: a) infecting a population ofcells with a library of vectors comprising polynucleotides encodingrecombinant the antibodies or fragments thereof that are displayed onthe extracellular surface of the cell membrane; and b) culturing thepopulation of cells under conditions that allow expression of theantibodies on the cell surface. 59 An antibody or fragment thereofcomprising an variant Fc region wherein said antibody has a reducedaffinity for an effector molecule. 60 The antibody or fragment thereofof embodiment 59, wherein the effector molecule is FcγRIIIA. 61 Theantibody or fragment thereof of embodiment 59, wherein the antibody hasreduced effector function. 62 The antibody or fragment thereof ofembodiment 61, wherein the effector function is ADCC. 63 The antibody orfragment thereof of embodiment 59 or 61, wherein the variant Fc regioncomprises at least one amino acid substitution, insertion or acombination thereof selected from the group consisting of: W277T;K246R/L251E/T260R; InR234/235; InV235/236; InR236/237; InR237/238;InV238/239; InN238/239; InL238/239; InE238/239; InG238/239; InS239/240;InG240/241; InE240/241; InG240/241/I198T; InL238/239/P238Q;InE238/239/V348A; InS239/240/V266A; InR237/238/G236A. 64 A kitcomprising: i) the library of embodiment 34. 65 The kit of embodiment64, further comprising a cell. 66 The kit of embodiment 65, wherein saidcell is a mammalian cell. 67 The kit of embodiment 66, wherein said cellis selected from the group consisting of NS0, CHO, Vero, Sf-9, COS7, 293or a derivative thereof.

8. EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples.

8.1 Example 1 Mammalian Cell Surface Display of Antibodies

The following sections describe the generation and characterization ofantibody fusion polypeptides that are efficiently displayed on thesurface of mammalian cells.

Polynucleotides encoding an immunoglobulin heavy chain fusionpolypeptide comprising a transmembrane domain and/or a GPI anchor signalare generated by PCR. Experiments described herein use the heavy chainof anti-EphA2 antibody 12G3H11 as an example unless otherwise noted.Representative examples of GPI anchor signal fusion partners are listedin Table 2. Polynucleotides encoding the various fusion proteins arecloned into pABHL, an antibody expression vector comprising anexpression cassette having the following operatively linked sequenceelements: 5′ end-CMV immediate early promoter-polynucleotide encodinglight chain signal peptide-polynucleotide encoding light chain variableregion-polynucleotide encoding kappa light chain constant region-ECMVIRES-polynucleotide encoding heavy chain leader peptide (sequencecomprises a unique XbaI restriction endonuclease recognitionelement)-polynucleotide encoding heavy chain variableregion-polynucleotide encoding IgG1 heavy chain constant region-uniqueNotI restriction endonuclease recognition element-Mo-MuLVIRES-polynucleotide encoding Neomycin resistance polypeptide-SV40polyA-3′ end. pABDisplay is a derivative of pABHL that further comprisesa DAF variant GPI anchor signal encoding polynucleotide operativelylinked to the 3′ end of the heavy chain constant region gene; thepABDisplay vector encodes an immunoglobulin heavy chain-DAF vGPI anchorsignal fusion polypeptide.

A polynucleotide encoding a heavy chain fusion polypeptide is generatedby PCR from a plasmid encoding the heavy chain gene of the 12G3H11anti-EphA2 antibody. The PCR reaction mix comprises a single forwardprimer UniXbaI (SEQ ID NO:) and multiple reverse primers. The UniXbaIprimer includes a recognition sequence for the XbaI restrictionendonuclease to facilitate cloning. The fusion partner specific reverseprimer sets were designed to include nucleotide residues encoding thefusion partner (e.g., a transmembrane domain) in frame with the heavychain, each set comprises multiple partially overlapping reverse primersof approximately 70 nucleotides. The first reverse primer of each setcomprises approximately 20 nucleotide residues at its 3′ end that annealto the DNA sequence encoding the heavy chain portion of the junctionbetween heavy chain and fusion partner. The second and subsequentreverse primers comprise ˜20 residues at their 3′ end that are identicalto the 5′ most ˜20 residues of the preceding reverse primer. The lastreverse primer comprises nucleotide residues encoding the C terminus ofthe fusion partner and a recognition sequence for the NotI restrictionendonuclease to facilitate subsequent cloning procedures. The sequenceof the three reverse primers used to generate the polynucleotidesequence encoding an anti-Eph2 heavy chain fused to a variant GPI anchorsignal of decay accelerated factor (DAF vGPI) are listed in Table 4. asan example for the primer design principles described above.

TABLE 4 Primers For Cell Surface Displayed Fusion Protein Generation.GPIDAFrev1 ACGGGTAGTACCTGAAGTGGTTCC (SEQ ID NO: 48)ACTTCCTTTATTTGGTTTACCCGG AGACAG GGAGAG GPIDAFrev2CAAACCTGTCAACGTGAAACACGT (SEQ ID NO: 49) GTGCCCAGATAGAAGACGGGTAGT ACCTGAAGTGGT GPIDAFrev3 TGAATTCGCGGCCGCTCAAGTCAG (SEQ ID NO: 50)CAAGCCCATGGTTACTAGCGT CC CAAGCAAACCTGTCAACGTGAAAC A

A PCR product of the expected size is digested with XbaI and Not I andligated into a similarly digested pABHL vector to facilitate expressionin a mammalian cell. The ligation product is used to transform DH10Bcompetent E. coli cells according to the manufacturer's protocols.Colonies of pABHL comprising the correct insert can be identified usingvarious methods known in the art (e.g. restriction digest of DNApreparation, diagnostic PCR amplification of test sequences); theiridentity can be further confirmed by sequencing using dideoxy sequencingreaction (e.g., BigDye® Terminator v3.0 Cycle Sequencing Ready ReactionKit, ABI). Plasmid DNA is prepared from selected clones using the QIAGENMini and Maxi Plasmid Kit according to the manufacturer's protocols.

HEK-293T cells are transiently transfected with a vector encoding ananti-EphA2 antibody fusion polypeptide tested. Transfected cells arecultured for approximately 24-48 hrs to allow antibody expression.

Cell surface display of an anti-EphA2 fusion antibody is detected bystaining the transfected cells with a FITC conjugated anti-human IgGantibody and analyzing them on a flow cytometer following standardprotocols. Shown in FIG. 2 (left side) are flow cytometry profilesobtained with cells expressing a DAF vGPI, CM GPI or thrombomodulin TMfused anti-EphA2 antibody.

The antigen binding characteristics of a cell surface displayedanti-EphA2 fusion antibody are ascertained by incubating the cells witha biotinylated EphA2-Fc fusion polypeptide. EphA2-Fc fusion proteinbound to the cell surface is visualized by further staining the cellswith FITC conjugated anti-biotin antibody and analyzing them on a flowcytometer following standard protocols. Shown in FIG. 2 (right side) areflow cytometry profiles obtained with cells expressing a DAF vGPI, CMGPI or thrombomodulin TM fused to the C terminus of the anti-EphA2antibody heavy chain.

8.2 Example 2 Fc Variant Library Construction

The following Example describes the generation of libraries comprisingFc variants of the anti-EphA2 antibody fused to DAF vGPI. Two differentlibraries were constructed: an Fc Substitution Library (SL-Fc) and an FcInsertion Library (IL-Fc).

A231, P232, E233, L242, K246, T250, L251, P257, V259, T260, C261, V273,K274, F275, W277, G281, and V282 with any one of the 19 other naturallyoccurring amino acids. For example, the substitution library comprisesFc regions having a substitution of the A residue at position 231 withan amino acid residue selected from the group consisting of G, L, M, F,W, K, Q, E, S, P, V, I, C, Y, H, R, N, D, and T. Individualsubstitutions are identified using standard nomenclature. For example anFc variant having a substitution of alanine (A) for glycine (G) atresidue 231 is identified as A231G.

Fc variants comprising the SL-Fc library are generated by PCR reactionsusing degenerate primers. Primers for SL-Fc generation are listed inTable 5. Separate sets of PCR reactions are used to generatepolynucleotides encoding heavy chains representing all possiblesubstitutions of each amino acid residues targeted in the FcSubstitution Library. For example, polynucleotides encoding thesubstitution mutants of residue A231 are generated by the followingthree PCR reactions: 1) 231A residue specific primer and MDAD-20universal primer are used to amplify the Fc region of DAF vGPI fusedanti-EphA2 heavy chain. 2) UniXbaI universal and 231A/232P/233Erevresidue specific primers are used to amplify the Fd region of DAF vGPIfused anti-EphA2 heavy chain. 3) PCR fragments from the first tworeactions are joined by overlap PCR using universal primers UniXbaI andMDAD-20. PCR fragments of the correct size representing all possiblesubstitution mutations of residue A231 are isolated from reaction 3,digested with XbaI and NotI restriction endonucleases and quantified.The SL-Fc library is generated by mixing an equimolar amount of PCRfragments representing each residue targeted and ligating the mixtureinto the pABDisplay vector comprising the light chain of 12G3H11anti-EphA2 antibody.

TABLE 5 Primers Used For Fc Substitution Library Generation. UniXbaI.GCT TGA GGT CTA GAC ATA TAT ATG GGT GAC AAT GAC ATC CAC TTT GCC TTT CTCTCC ACA GGT GTC CAC TCC (SEQ ID NO: 10) MDAD-20 AAC CTC TAC AAA TGT GGTATG GCT (SEQ ID NO: 11) 231Afor ACA TGC CCA CCG TGC CCA NNS CCT GAA CTCCTG GGG GGA (SEQ ID NO: 12) 232Pfor ACA TGC CCA CCG TGC CCA GCA NNS GAACTC CTG GGG GGACCG (SEQ ID NO: 13) 233Efor ACA TGC CCA CCG TGC CCA GCACCT NNS CTC CTG GGG GGA CCG TCA (SEQ ID NO: 14) 231A/232P/233Erev TGGGCA CGG TGG GCA TGT (SEQ ID NO: 15) 242Lfor GGG GGA CCG TCA GTC TTC NNSTTC CCC CCA AAA CCC AAG (SEQ ID NO: 16) 246Kfor GGG GGA CCG TCA GTC TTCCTC TTC CCC CCA NNS CCC AAG GAC ACC CTC ATG (SEQ ID NO: 17) 2421/246KrevGAA GAC TGA CGG TCC CCC (SEQ ID NO: 18) 250Tfor CCC CCA AAA CCC AAG GACNNS CTC ATG ATC TCC CGG ACC (SEQ ID NO: 19) 251Lfor CCC CCA AAA CCC AAGGAC ACC NNS ATG ATC TCC CGG ACC CCT (SEQ ID NO: 20) 250T/251Lrev GTC CTTGGG TTT TGG GGG (SEQ ID NO: 21) 257Pfor CTC ATG ATC TCC CGG ACC NNS GAGGTC ACA TGC GTG GTG (SEQ ID NO: 22) 259Vfor CTC ATG ATC TCC CGG ACC CCTGAG NNS ACA TGC GTG GTG GTG GAC (SEQ ID NO: 23) 260Tfor CTC ATG ATC TCCCGG ACC CCT GAG GTC NNS TGC GTG GTG GTG GAC GTG (SEQ ID NO: 24) 261CforCTC ATG ATC TCC CGG ACC CCT GAG GTC ACA NNS GTG GTG GTG GAC GTG AGC (SEQID NO: 25) 257P/259V/260T/261Crev GGT CCG GGA GAT CAT GAG (SEQ ID NO:26) 273Vfor AGC CAC GAA GAC CCT GAG NNS AAG TTC AAC TGG TAC GTG (SEQ IDNO: 27) 274Kfor AGC CAC GAA GAC CCT GAG GTC NNS TTC AAC TGG TAC GTG GAC(SEQ ID NO: 28) 275Ffor AGC CAC GAA GAC CCT GAG GTC AAG NNS AAC TGG TACGTG GAC GGC (SEQ ID NO: 29) 277Wfor AGC CAC GAA GAC CCT GAG GTC AAG TTCAAC NNS TAC GTG GAC GGC GTG GAG (SEQ ID NO: 30) 273V/274K/275F/277WrevCTC AGG GTC TTC GTG GCT (SEQ ID NO: 31) 281Gfor TTC AAC TGG TAC GTG GACNNS GTG GAG GTG CAT AAT GCC (SEQ ID NO: 32) 282Vfor TTC AAC TGG TAC GTGGAC GGC NNS GAG GTG CAT AAT GCC AAG (SEQ ID NO: 33) 284Vfor TTC AAC TGGTAC GTG GAC GGC GTG GAG NNS CAT AAT GCC AAG ACA AAG (SEQ ID NO: 34)281G/282V/284Vrev GTC CAC GTA CCA GTT GAA (SEQ ID NO: 35)

The Fc Insertion Library (IL-Fc) comprises variant Fc regions having aninsertion of a single amino acid residue between amino acid residues 230and 231, 231 and 232, 232 and 233, 233 and 234, 234 and 235, 235 and236, 236 and 237, 237 and 238, 238 and 239, 239 and 240, or 240 and 241wherein the inserted residue may comprise any one of the twentynaturally occurring amino acids. Individual insertions are identified as“In” followed by the one letter code of the inserted amino acid residueand the position of the residues immediately flanking the insertion. Forexample InG231/232 denotes a variant Fc comprising an insertion of aglycine between residues 231 and 232.

Fc variants comprising the IL-Fc library are generated by PCR reactionsusing degenerate primers. Primers for IL-Fc generation are listed inTable 6. Separate sets of PCR reactions are used to generatepolynucleotides encoding heavy chains representing all possible aminoacid insertions at each of the positions targeted in the Fc InsertionLibrary. For example, polynucleotides encoding the insertion mutants atposition 230/231 are generated by the following three PCR reactions: 1)Position specific primer 230/231 Infor and universal primer MDAD-20 areused to amplify the Fc region of DAF vGPI fused anti-EphA2 heavy chain.2) Universal primers UniXbaI and Inrev are used to amplify the Fd regionof DAF vGPI fused anti-EphA2 heavy chain. 3) PCR fragments from thefirst two reactions are joined by overlap PCR using universal primersUniXbaI and MDAD-20. PCR fragments of the correct size representing allpossible insertion mutations of position 230/231 are isolated fromreaction 3, digested with XbaI and NotI restriction endonucleases andquantified. The IL-Fc library is generated by mixing an equimolar amountof PCR fragments representing each targeted position and ligating themixture into the pABDisplay vector comprising the light chain of 12G3H11anti-EphA2 antibody.

TABLE 6 Primers Used For Fc Insertion Library Generation. 230/231InforACA TGC CCA CCG TGC CCA NNS GCA CCT GAA CTC CTG GGG (SEQ ID NO: 36)231/232Infor ACA TGC CCA CCG TGC CCA GCA NNS CCT GAA CTC CTG GGG GGA(SEQ ID NO: 37) 232/233Infor ACA TGC CCA CCG TGC CCA GCA CCT NNS GAA CTCCTG GGG GGA CCG (SEQ ID NO: 38) 233/234Infor ACA TGC CCA CCG TGC CCA GCACCT GAA NNS CTC CTG GGG GGA CCG TCA (SEQ ID NO: 39) 234/235Infor ACA TGCCCA CCG TGC CCA GCA CCT GAA CTC NNS CTG GGG GGA CCG TCA GTC TTC CTC (SEQID NO: 40) 235/236Infor ACA TGC CCA CCG TGC CCA GCA CCT GAA CTC CTG NNSGGG GGA CCG TCA GTC TTC CTC TTC (SEQ ID NO: 41) 236/237Infor GCA CCT GAACTC CTG GGG NNS GGA CCG TCA GTC TTC CTC (SEQ ID NO: 42) 237/238Infor GCACCT GAA CTC CTG GGG GGA NNS CCG TCA GTC TTC CTC TTC (SEQ ID NO: 43)238/239Infor GCA CCT GAA CTC CTG GGG GGA CCG NNS TCA GTC TTC CTC TTC CCC(SEQ ID NO: 44) 239/240Infor GCA CCT GAA CTC CTG GGG GGA CCG TCA NNS GTCTTC CTC TTC CCC CCA (SEQ ID NO: 45) 240/241Infor GCA CCT GAA CTC CTG GGGGGA CCG TCA GTC NNS TTC CTC TTC CCC CCA AAA (SEQ ID NO: 46) Inrev CCCCAG GAG TTC AGG TGC (SEQ ID NO: 47)

8.3 Example 3 Streptavidin Fused FcγRIIIA Reagent

Primer pair SA1/SA2 (see Table 7.) is used to PCR amplify polynucleotideS encoding streptavidin from template genomic DNA of Streptomycesavidinii. Primer pair A1/A2 (see Table 7.) is used to PCR amplifypolynucleotide F encoding the extracellular domain of FcγRIIIA from ahuman bone marrow cDNA library (clontech) template. Overlapping PCRexploiting the partial sequence complementarity of primers A2 and SA1 isused to generate polynucleotides FA encoding a FcγRIIIA-streptavidinfusion polypeptide. NcoI/NheI digested polynucleotide FA is cloned intothe pET-28a (Novagen) expression vector and FcγRIIIA-streptavidin fusionpolypeptide is expressed in bacteria following the manufacturer'sinstructions. Recombinant FcγRIIIA-streptavidin fusion protein isrecovered from inclusion bodies and refolded as described by Gao, et al.(1997, Proc Natl Acad Sci USA. 94:11777-82). The refolded fusion proteinis subsequently purified on an immunobiotin column (PIERCE) according tomanufacturer's instructions. The final concentration of theFcγRIIIA-streptavidin preparation is approximately 2.4 mg/ml.

TABLE 7 PCR Primers For Amplifying Streptavidin And The ExtracellularDomains Of FcγRIIIA. A1 primer AAGCTTCGGTCCG CCACCATGGCAACTGAAGATCTCCCAAAG (SEQ ID NO: 51) A2 primer GTCTGCCGAACCGCTGCCTGCCAAACCTTGAGTGATGGT(SEQ ID NO: 52) SA1 GGCAGCGGTTCGGCAGACCCCTCCAAGGAC primer (SEQ ID NO:53) SA2 CAGGGGCTAGCTTACTGCTGAACGGCGTCGAGCGG primer (SEQ ID NO: 54)

8.4 Example 4 Selection of Fc Variants with Altered FcγRIIIA BindingProperties

Control experiments are performed to optimize the conditions fortransient transfection with Lipofectamine™ 2000 (Invitrogen). HEK-293cells are transfected with different amounts (e.g., 0.05, 0.1, 0.5, 1.0,2.0, 4, and 10 μg) of a pABDisplay vector expressing an anti-EphA2antibody fused to the DAF vGPI signal sequence. The expression level ofanti-EphA2 antibody-DAF vGPI fusion polypeptide is ascertained bycontacting the transfected cells with FcγRIIIA-streptavidin fusionprotein followed by staining with FITC conjugated anti-streptavidinantibody. The cells are subsequently analyzed with a flow cytometer. Areproducible shift in fluorescence intensity is seen for each vectoramount tested. The largest shift is observed for plasmid amounts of 4 μgand above. Representative flow cytometry profiles are shown in FIG. 3.

Control experiments are performed to optimize the conditions for a cellsurface FcγRIIIA binding assay. HEK-293 cells are transfected with 10 μgof a pABDisplay vector expressing a DAF GPI signal fused anti-EphA2antibody. Separate aliquots of the transfected cells are contacted with1:500, 1:1000, 1:2000, 1:3000, 1:4000 or 1:5000 fold dilutedFcγRIIIA-streptavidin fusion protein followed by staining with FITCconjugated anti-streptavidin antibody. Cells are subsequently analyzedwith a flow cytometer. While each concentration of FcγRIIIA-streptavidinfusion protein used results in a shift of fluorescence intensity, theshifts are less pronounced at dilutions of FcγRIIIA-SA above 1:1000.Representative of flow cytometry profiles are shown in FIG. 4.

Transient transfection of an Fc variant library: HEK-293 cells (6×10⁶cells) in 12 ml of growth medium are plated in 100×20 mm tissue cultureplates the day before transfection. On the day of transfection, 0.5-10μg of Fc mutant library plasmid is mixed with 30 μl of Lipofectamine2000 in OPTI-MEM medium and added into the medium of HEK-293 cells.After 48 hrs incubation post-transfection at 37° C. the transfectedcells are detached by using Accutase enzyme cell detachment medium(Chemicon) and washed with cold FACS buffer (PBS/10% FBS). Cells areresuspended in 200 μl of FACS buffer containing 1:500 to 1:5000 dilutedrecombinant FcγRIIIA-streptavidin fusion protein and incubated for 20min at RT. Cells are washed again with FACS buffer and stained with FITCconjugated anti-streptavidin antibody for 20 min at RT followingstandard protocols. Cells are washed to remove any unboundanti-streptavidin antibody and re-suspended at a density of 2×10⁶/ml.

Isolation of cells expressing Fc variants with altered affinity forFcγRIIIA: Resuspended cells are analyzed on a flow cytometer. Arepresentative example of a fluorescent staining profile is shown inFIG. 5A. Cells with very low or very high fluorescence intensity can beisolated via FACS. An example of the gates suitable for sorting cellswith very low staining is shown in FIG. 5A. Isolated cells arere-analyzed on the flow cytometer to check the quality of the sort (FIG.5B).

Recovery of transiently transfected library DNA: Sorted cells arecollected by centrifugation and resuspended in 0.4 ml of cell lysissolution (0.6% SDS and 10 mM EDTA). After 20 min incubation at RT 100 μlof 5 M Nacl is added to the cell lysate. Cell lysate is cleared bycentrifugation and the supernatant is extracted withphenol/chloroform/isoamylalcohol (25:24:1). DNA is precipitated withethanol from the aqueous fraction. DH10B E. coli cells are transformedby electroporation with half of the recovered DNA and plated on LB agarplate containing 100 μg/ml carbencilline. After overnight growth, all ofthe bacterial cells are scraped off the agar plate and used for plasmidDNA extraction.

Additional round(s) of selection: The plasmid DNA recovered may besubjected to additional round(s) (e.g., a total of three rounds) of theabove described selection process to further enrich for clones encodingan Fc variant with altered FcγRIIIA binding affinity.

8.5 Example 5 Initial Characterization of Isolated Fc Variants

Fc variants isolated using the selection procedure described in Example4 are initially characterized as follows. After approximately threerounds of selection, DH10B E. coli cells are transformed with DNArecovered from the sorted cell population, individual bacterial clonesare selected and plasmid DNA is isolated following standard protocols.

HEK-293 cells are transfected with the isolated plasmid DNA. An aliquotof the transfected cells are stained with FITC conjugated anti-humanIgG(H+L) antibody and analyzed on a flow cytometer to ascertain the cellsurface expression level of the Fc variant. Examples of stainingprofiles are shown in FIG. 6A, B, and C.

The FcγRIIIA binding affinity of the isolated Fc variant is alsodetermined. A separate aliquot of transfected cells are incubated withrecombinant FcγRIIIA-streptavidin fusion protein followed by stainingwith FITC conjugated anti-streptavidin antibody as described in Example3. The staining profile of the cells is determined using a flowcytometer. Examples of staining profiles are shown in FIG. 6D, E, and F.

Fc variant clones with staining profile indicative of high cell surfaceexpression and low FcγRIIIA affinity are selected for furthercharacterization. An example of such a clone is Fc variant InR236/237shown in FIG. 6C and F.

Example 6 Mammalian Expression of Soluble Fc Variants

To express a soluble Fc variant, an oligonucleotide encoding the Fcvariant without the DAF vGPI signal sequence is generated by PCR usingthe UniXbaI and BackNotI (BackNotI:TCAATGAATTCGCGGCCGCTCATTTACCCGGAGACAGGGAGAGGC (SEQ ID NO:55)) primers.The PCR product of expected size is digested with XbaI and Not Irestriction endonucleases and ligated into an XbaI NotI cleaved pABHLexpression vector comprising the light chain of 1 2G3H11 anti-EphA2antibody. Bacterial clones having the correct expression construct canbe identified using various methods known in the art (e.g. restrictiondigest of vector DNA preparation, diagnostic PCR amplification of vectorsequences). The identity of the clones can be further confirmed bysequencing using the dideoxy method (e.g., BigDye® Terminator v3.0 CycleSequencing Ready Reaction Kit, ABI). Plasmid DNA is prepared fromselected clones using the QIAGEN Mini and Maxi Plasmid Kit according tothe manufacturer's protocols

HEK-293 cells are transfected with a pABHL vector comprising apolynucleotide encoding a soluble Fc variant using Lipofectamine™ 2000(Invitrogen) transfection reagent. Transfected cells are incubated fornine days to allow for Fc variant production. Conditioned medium iscollected on day 3, 6, and 9 of the incubation period. The Fc variant ispurified using a pre-cast protein A column (GE Healthcare). The bound Fcvariant is eluted from the column with low pH buffer, neutralized, anddialyzed against PBS. The concentration of the purified Fc variant iscalculated from the solution's optical density at 280 nm.

8.6 Example 7 Fc Binding Assays

Fc variants isolated using the methods described above are assayed fortheir binding affinity to one or more isolated Fc receptors and/or Fcligand (e.g., FcγRIIIA, C1q) in an ELISA assay format. ELISA assays areperformed following standard protocols. Commercially available reagentsare used according to the manufacturer's instructions.

Microtiter plates are coated with protein A/G (PIERCE) solution (0.25μg/ml) and incubated at 4° C. overnight. Any remaining binding sites areblocked with 4% skimmed milk in PBS buffer (blocking buffer) for 1 h at37° C. Approximately 25-50 μl of control, wild type or Fc variant mutantantibody solution is added to each well and incubated for 1 h at 37° C.After washing the wells, FcγRIIIA-streptavidin fusion protein (1:1000dilution in 1% BSA) is added for 1 hour at 37° C., followed by washingand incubation with biotin-conjugated HRP for 30 min. Detection iscarried out by adding 30 μl of tetramethylbenzidine (TMB) substrate(Pierce) followed by neutralization with 30 μl of 0.2 M H₂SO₄. Theabsorbance is read at 450 nm. IC50 values may be determined andnormalized to those obtained for a wild type antibody control assayed atthe same time (e.g., in the same microtiter plate). Examples of bindingcurves for wild type and several Fc variants are shown in FIG. 7.

Microtiter plates coated with protein A/G (PIERCE) solution (0.25 μg/ml)are incubated at 4° C. overnight. Any remaining binding sites areblocked with blocking buffer for 1 h at 37° C. Approximately, 25-50 μlper well of wild type or Fc variant mutant antibody solution is added toeach well and incubated for 1 h at 37° C. . After washing the wells,approximately 100 μl of 2 μg/ml human C1q (Quidel, Calif.) is added for1 h at 37° C. After washing the wells, they are incubated with sheepanti-human C1q antibody (BioDesign) for 1 h at 37° C. After another washthe wells are incubated with a horseradish peroxydase conjugated donkeyanti-sheep IgG (Serotec, NC) for 1 h at room temperature. Horseradishperoxydase activity is detected with TMB substrate (KPL, MD). Thereaction is quenched with 0.2 M H₂SO₄. The absorbance is read at 450 nm.IC50 values are determined and may be normalized to those obtained for awild type antibody control assayed at the same time (e.g., in the samemicrotiter plate). Examples of binding curves for wild type and severalFc variants are shown in FIG. 8.

Representative binding curves of several Fc variants toFcγRIIIA-streptavidin and C1q are shown in FIGS. 7 and 8, respectively.The anti-EphA2 wild type antibody and an IgG4 isotype control antibodyare used as positive and negative controls, respectively, for each assayperformed. Each of the Fc variants assayed has a reduced binding toFcγRIIIA and C1q compared to the wild type antibody. The data for theseFc variants and others are summarized in Table 8.

Example 8 Cell Surface Receptor Binding Assays

Soluble Fc variants isolated using the methods described above areassayed for their binding affinity to the surface of cells expressingone or more Fc receptors (e.g., FcγRIIIA, FcγRII).

Two cell types are utilized, THP-1 cells which predominantly expressFcγRII with a small amount of FcγRI present on the cell surface and NKcells which express FcγRIIIA almost exclusively. Early passage THP-1cells are used and NK cells are isolated from healthy donors by using anNK cell isolation kit from Miltenyi Biotec. For binding of Fc variantsto Human NK cell surface (FcγRIIIA), ˜10 μl of the Fc variant atdifferent concentrations (e.g., 10 μg/ml to 1 μg/ml) is added to thecells and incubated at 4° C. for 30 min. The cells are washed with FACSbuffer, then stained with FITC conjugated goat anti-human IgG(H+L) Fab(Pierce) for 30 min at 4° C. The cells are then washed and analyzed byGuava EasyCyte cytometer. For binding of Fc variants to THP-1 cellsurface (FcγRI and FcγRII), ˜10 μl of the Fc variant at differentconcentrations (e.g., 10 μg/ml to 1 μg/ml) is added to the cells,incubated at 4° C. for 30 min. The cells are washed with FACS buffer,then stained with FITC conjugated goat ant-human IgG(H+L) Fab (Pierce)for 30 min at 4° C. The cells are then washed and analyzed by GuavaEasyCyte cytometer.

The percentage of THP-1 and NK cells bound by several Fc variants areshown in FIGS. 9 and 10, respectively. The anti-EphA2 wild type antibodyand an IgG4 isotype control antibody are used as positive and negativecontrols, respectively, for each assay performed. Each of the Fcvariants assayed has a reduced binding to the cell surface receptorspresent on THP-1 and NK cells compared to the wild type antibody. Thedata for these Fc variants and others are summarized in Table 8.

Example 9 Antigen Binding

Antigen binding may be determined using methods well known in the art.For example an ELISA based assay following standard protocols may beused. Briefly, Microtiter plates are coated with protein A/G (PIERCE)solution (0.25 μg/ml) and incubated at 4° C. overnight. The plates arethen washed with PBS/0.1% Tween-20 and any remaining binding sites areblocked with blocking buffer. 50 μl of test antibody at concentrationsfrom ˜5000 ng/ml to ˜5 ng/ml, are added to each well and incubated for˜60 min at 37° C. ˜50 μl of an appropriate dilution of biotin conjugatedEphA2 protein (e.g., EphA2-Fc fusion described in Dall'Acqua, F. M. etal., J Immunol, 177: 1129-1138 (2006)) is added to each well andincubated for ˜60 min at 37° C., followed by washing. Horseradishperoxidase conjugated streptavidin is added to each well and incubatedfor 30 min at 37° C. following the manufacturer's instructions.Detection is carried out by adding 30 μl of tetramethylbenzidine (TMB)substrate (Pierce) followed by neutralization with 30 μl of 0.2 M H₂SO₄.The absorbance is read at 450 nm.

The ligand binding activity of several Fc variants that showed reducedFc receptor binding and/or ADCC activity was examined by ELISA. The wildtype anti-EphA2 antibody and two antibodies of irrelevant specificity(Vitaxin and anti-HMBG1) were used as positive and negative controls,respectively, for each assay. The antibodies were tested by ELISA assayusing a biotinylated human EphA2 protein. All of the Fc variantsexamined showed binding affinity for human EphA2 similar to that of thewild type antibody; the IC50 values are indicated (FIG. 11).

Example 10 Antibody Dependent Cell Mediated Cytotoxicity (ADCC) Assay

Antibody dependent cell cytotoxicity (ADCC) is assayed in a four-hournon-radioactive lactate dehydrogenase (LDH) release assay (PromegaCorporation, Madison, Wis.). Briefly, EphA2 expressing A549 target cellsare distributed into 96-well U-bottomed plates (1×10⁴/50 μl) andpre-incubated with serial dilution of antibodies (50 μl) for 20 min at37° C. Human effector cells (100 μl) are then added at effector totarget cell ratios of 50:1 and 25:1. Peripheral blood mononuclear cells(PBMC) purified from healthy human donors using Lymphocyte SeparationMedium (MP Biomedicals, Irvine, Calif.), resuspended in the medium(RPMI-1640 10% FBS—2 mM L-Glu-Pen/Strep, 5 ng/ml of IL-2), and incubatedat 37° C. for overnight were used as effector cells. After 4 hrs ofincubation at 37° C., plates are centrifuged, and cell death is analyzedby measuring the release of LDH into the cell supernatant with a30-minute coupled enzymatic assay. The percentage of specific lysis iscalculated according to the formula: % specificlysis=100×(EX−ESpon−TSpon)/(Tmax−Tspon), where EX represents the releasefrom experimental wells, Espon is the spontaneous release of effectorcells alone, Tspon is spontaneous release of target cells alone, andTmax is the maximum release from lysed target cells.

Shown in FIG. 12 are cytotoxicity curves from representative ADCC assaysperformed using several of the isolated Fc variants. A positive controlwild type anti-EphA2 antibody and a negative control anti-CD4 antibody(R347) are also included in the assay. A549 cells expressing EphA2, butnot CD4, are used as targets. Effector cells are purified from healthyhuman donors. The assays are performed using two different ratios oftarget to effector cell (50:1 and 25:1) and antibody concentrationsranging from 0.1 to 10000 ng per well. Each of the Fc variants and thenegative control have little to no activity above background while thewild type antibody mediates efficient lysis of the target cells at bothtarget to effector ratios. The results for a number of Fc variants aresummarized in Table 8.

TABLE 8 Binding Affinity and ADCC Activity of Fc Variants FcγRIIIA C1qFcγRI&II ADCC Fc mutants Binding binding Binding activity Wild type 0.15ug/ml  W277T  0.5 ug/ml ND ND ND K246R/L251E/ >20 ug/ml 2 fold les  9fold less not induce T260R InR236/237 >20 ug/ml 2 fold les 20 fold lessnot induce InN238/239 >20 ug/ml 5 fold less 20 fold less not induceInE240/241 4.927 ug/ml   ND ND InV238/239 >20 ug/ml 2 fold less  5 foldless not induce InG240/241 + >20 ug/ml 4 fold less 20 fold less notinduce I198T InR234/235 >20 ug/ml 3 fold less 20 fold less not induceInL238/239 + >20 ug/ml 4 fold less  9 fold less not induce P238QInE238/239 + >20 ug/ml 6 fold less  6 fold less not induce V348AInS239/240 + >20 ug/ml 3 fold less 27 fold less not induce V266AInR237/238 + >20 ug/ml 5 fold less 27 fold less not induce G236A

Example 11 cDNA Library Synthesis

First, total RNA is isolated from the peripheral blood mononuclear cells(PBMC) of twelve healthy donors e.g., by using QIAgen RNeasy kit. Inaddition, a pool of mRNA is obtained by combining material from severalsources (Bioscience, Cat# 636170, BD Bioscience Cat. 6594-1, Origenetechnologies and Biochain Institute, Inc. Cat#M 1234246). A human cDNAlibrary is synthesized by using Superscript III RT kit (Invitrogen)following the manufacturer's instructions.

Example 12 pENABdisplay Vector Construction

The pENTR™2B (Invitrogen) is digested with XbaI and SfoI to delete theccdB gene. The larger fragment from the Spel and BSTZ17I digested12G3H11 pABdisplay vector comprising the antibody expression cassettewas cloned into the XbaI SfoI digested pENTR™2B vector. The resultingvector, designated as pENPABdisplay, comprises a 12G3H11 anti-EphA2-DAFvGPI fusion antibody expression cassette flanked by the attL1 and attL2recombination signals.

Example 13 pENABdisplay Heavy Chain Library Construction

Rearranged VH segments are PCR amplified from a human cDNA library (seeExample 11). Primers used are listed in Table 9. PCR reactions toamplify polynucleotides encoding a signal sequence are performed in avolume of 100 μl containing 40 ng of pENABdisplay, 10 pmol of theHcldf-forward and 84830-D10-reverse primers, and Pfu ultra Taqpolymerase (Stratagene, Cat. 600380) following the manufacturer'ssuggestion. The PCR reaction is initially heated to 95° C. for 5minutes,followed by 25 cycles of 95° C. for 30 sec, 55° C. for 30 sec, 68° C.for 45 sec and held at 68° C. for 7 minutes. The PCR product purifiedfor example by using the QIAgen PCR purification Kit (Cat. 28106). Theheavy chain variable regions are amplified separately from the cDNAlibrary using Taq DNA polymerase (Invitrogen, cat. 18038-018), 50 pmolof Medieu-VH1-15 and 50 pmol of the pooled reverse-Medieu-JH1, JH2 andJH3 primers following the manufacturer's instructions. After 5 minutesof denaturing, the template is amplified for 8 cycles at 95° C. for 30sec, 52° C. for 60 sec and 72° C. for 60 sec; the template is furtheramplified for 32 cycles at 95° C. for 30 sec, 62° C. for 30 sec, 72° C.for 60 sec and held at 72° C. for 7 minutes. The VH fragments agarosegel purified and an overlapping PCR is performed with the VH fragmentsand the signal sequence using the forward-HcldF and pooledreverse-medieu-JH 1, JH2 and JH3 primers; the PCR reaction is performedwith Taq DNA polymerase, 20 ng of each template and 50 pmol of theprimers. After 5 minutes of denaturing, the template is amplified for 8cycles without the primers at 95° C. for 30 sec, 55° C. for 45 sec and68° C. for 60 sec; the template is further amplified for 25 cycles withprimers at 95° C. for 30 sec, 55° C. for 30 sec, 68° C. for 60 sec andheld at 72° C. for 7 minutes. The PCR product is gel purified asdescribed previously. An equal amount of each product is mixed anddigested with XbaI and SalI restriction endonucleases (New EnglandBiolabs) and cloned into the pENPABdisplay vector to create the heavychain (IgG1) library. The library degree of diversity is determined bysequencing 96 clones.

TABLE 9 Primers used for human naïve antibody library generation. HumanV heavy specific forward primers Medieu-VH1GCCTTTCTCTCCACAGGTGTACACTCCCAGGTKCAGCTG GTGCAGTGTGG (SEQ ID NO: 63)Medieu-VH2 GCCTTTCTCTCCACAGGTGTACACTCCCAGGTCCAGCTT GTGCAGTCTGG (SEQ IDNO: 64) Medieu-VH3 GCCTTTCTCTCCACAGGTGTACACTCCSAGGTCCAGCTG GTACAGTCTGG(SEQ ID NO: 65) Medieu-VH4 GCCTTTCTCTCCACAGGTGTACACTCCCARATGCAGCTGGTGCAGTCTGG (SEQ ID NO: 66) Medieu-VH5GCCTTTCTCTCCACAGGTGTACACTCCCAGATCACCTTG AAGGAGTCTGG (SEQ ID NO: 67)Medieu-VH6 GCCTTTCTCTCCACAGGTGTACACTCCCAGGTCACCTTG AAGGAGTCTGG (SEQ IDNO: 68) Medieu-VH7 GCCTTTCTCTCCACAGGTGTACACTCCGARGTGCAGCTG GTGGAGTCT(SEQ ID NO: 69) Medieu-VH8 GCCTTTCTCTCCACAGGTGTACACTCCCAGGTGCAGCTGGTGGAGTCTGG (SEQ ID NO: 70) Medieu-VH9GCCTTTCTCTCCACAGGTGTACACTCCGAGGTGCAGCTG TTGGAGTCTGG (SEQ ID NO: 71)Medieu-VH10 GCCTTTCTCTCCACAGGTGTACACTCCGAGGTGCAGCTG GTGCAGWCYGG (SEQ IDNO: 72) Medieu-VH11 GCCTTTCTCTCCACAGGTGTACACTCCCAGSTGCAGCTG CAGGAGTCSGG(SEQ ID NO: 73) Medieu-VH12 GCCTTTCTCTCCACAGGTGTACACTCCCAGGTGCAGCTACAGCAGTGGGG (SEQ ID NO: 74) Medieu-VH13GCCTTTCTCTCCACAGGTGTACACTCCGARGTGCAGCTG GTGCAGTCCTGG (SEQ ID NO: 75)Medieu-VH14 GCCTTTCTCTCCACAGGTGTACACTCCCAGGTACAGCTG CAGCAGTCAGG (SEQ IDNO: 76) Medieu-VH15 GCCTTTCTCTCCACAGGTGTACACTCCCAGGTGCAGCTG GTGCAATCTGG(SEQ ID NO: 77) Human V heavy specific reverse primers Medieu-JH1GAAGACGGATGGGCCCTTGGTCGACGCTGAGGAGACRGT GACCAGGGT (SEQ ID NO: 78)Medieu-JH2 GAAGACGGATGGGCCCTTGGTCGACGCTGAAGAGACGGT GACCATTGT (SEQ ID NO:79) Medieu-JH3 GAAGACGGATGGGCCCTTGGTCGACGCTGAGGAGACGGT GACCGTGGT (SEQ IDNO: 80) Human V kappa specific forward primers Medieu-VK1CTCTGGCTCCCCGGGGCGCGCTGTRACATCCAGATGACC CAGTCTCC (SEQ ID NO: 81)Medieu-VK2 CTCTGGCTCCCCGGGGCGCGCTGTGMCATCCRGWTGACC CAGTCTCC (SEQ ID NO:82) Medieu-VK3 CTCTGGCTCCCCGGGGCGCGCTGTGTCATCTGGATGACC CAGTCTCC (SEQ IDNO: 83) Medieu-VK4 CTCTGGCTCCCCGGGGCGCGCTGTGATATTGTGATGACC CAGACTCC (SEQID NO: 84) Medieu-VK5 CTCTGGCTCCCCGGGGCGCGCTGTGATRTTGTGATGACW CAGTCTCC(SEQ ID NO: 85) Medieu-VK6 CTCTGGCTCCCCGGGGCGCGCTGTGAAATTGTGTTGACRCAGTCTCC (SEQ ID NO: 86) Medieu-VK7CTCTGGCTCCCCGGGGCGCGCTGTGAAATAGTGATGACG CAGTCTCC (SEQ ID NO: 87)Medieu-VK8 CTCTGGCTCCCCGGGGCGCGCTGTGAAATTGTAATGACA CAGTCTCC (SEQ ID NO:88) Medieu-VK9 CTCTGGCTCCCCGGGGCGCGCTGTGACATCGTGATGACC CAGTCTCC (SEQ IDNO: 89) Medieu-VK10 CTCTGGCTCCCCGGGGCGCGCTGTGAAACGACACTCACG CAGTCTCC(SEQ ID NO: 90) Medieu-VK11 CTCTGGCTCCCCGGGGCGCGCTGTGAAATTGTGCTGACTCAGTCTCC (SEQ ID NO: 91) Human V kappa specific reverse primers CkappaGCATGCTCGACATCGATTCACTAACACTCTCCCCTGTTG AAGCTC (SEQ ID NO: 92) Human Vlambda specific forward primers Medieu-V?1CTCTGGCTCCCCGGGGCGCGCTGTCAGTCTGTGCTGACT CAGCCACC (SEQ ID NO: 93)Medieu-V?2 CTCTGGCTCCCCGGGGCGCGCTGTCAGTCTGTGYTGACG CAGCCGCC (SEQ ID NO:94) Medieu-V?3 CTCTGGCTCCCCGGGGCGCGCTGTCAGTCTGCCCTGACT CAGCCT (SEQ IDNO: 95) Medieu-V?4 CTCTGGCTCCCCGGGGCGCGCTGTTCCTATGWGCTGACW CAGCCA (SEQID NO: 96) Medieu-V?5 CTGTGGCTCCCCGGGGCGCGCTGTTCCTATGAGCTGACA CAGCTACC(SEQ ID NO: 97) Medieu-V?6 CTCTGGCTCCCCGGGGCGCGCTGTTCTTCTGAGCTGACTCAGGACC (SEQ ID NO: 98) Medieu-V?7CTCTGGCTCCCCGGGGCGCGCTGTTCCTATGAGCTGATG CAGCCAC (SEQ ID NO: 99)Medieu-V?8 CTCTGGCTCCCCGGGGCGCGCTGTCAGCYTGTGCTGACT CAATC (SEQ ID NO:100) Medieu-V?9 CTCTGGCTCCCCGGGGCGCGCTGTCWGSCTGTGCTGACT CAGCC (SEQ IDNO: 101) Medieu-V?10 CTCTGGCTCCCCGGGGCGCGCTGTAATTTTATGCTGACT CAGCCCCA(SEQ ID NO: 102) Medieu-V?11 CTCTGGCTCCCCGGGGCGCGCTGTCAGRCTGTGGTGACYCAGGAGCC (SEQ ID NO: 103) Medieu-V?12CTCTGGCTCCCCGGGGCGCGCTGTCAGGCAGGGCTGACT CAGCCACC (SEQ ID NO: 104) HumanVlambda specific reverse primers Clambda1GCATGCTCGACATCGATTCACTATGAACATTCTGTAGGG GCCACTG (SEQ ID NO: 105)Clambda2 GCATGCTCGACATCGATTCACTAAGAGCATTCTGCAGGG GCCACTG (SEQ ID NO:106) V heavy signal sequence specific primers HcldFCCATGGGATGGAGCTGTATCA (SEQ ID NO: 107) 84830-D10GGAGTGTACACCTGTGGAGAGAAAGGC (SEQ ID NO: 108)

Example 14 pENABdisplay Light Chain Library Construction

Rearranged antibody kappa and lambda light chain segments are PCRamplified from a human cDNA library (see Example 11). Primers used arelisted in Table 9. Twelve VHλ forward primers are paired with two λreverse primers to amplify the antibody λ light chain variable andconstant regions. Similarly, eleven VHκ forward primers are paired withthe κ reverse primer to amplify the antibody κ light chain variable andconstant regions. Using Pfu Ultra (Stratagene) and following themanufacture's instructions, each reaction is done separately using 10pmol of each primer. After the initial 3 minutes denaturation, the PCRreaction is amplified for 30 cycles at 95° C. for 30 sec, 52° C. for 30sec, 68° C. for 90 sec and held at 68° C. for 10 minutes. The PCRproducts are pooled, agarose gel purified and digested with BssHII andC1aI restriction endonucleases. Using similarly digested pENABdisplayvector, the products are T4 DNA ligated, phenol-chloroform extracted,precipitated and transformed into DH10B electrocompetent cells. Thelibrary's degree of diversity is determined by sequencing 96 clones.

Example 15 pENABdisplay Heavy Chain-Light Chain Library Construction

To combine the diverse antibody heavy chains and light chains of theheavy chain and light chain libraries, respectively, into a single heavychain-light chain library, the pENPABdisplay light chain library isdigested with Xba I and Not I restriction endonucleases. ThepENPABdisplay heavy chain library is similarly digested to release thediverse heavy chain encoding fragments, which are then agarose gelpurified and ligated into the XbaI NotI digested pENABdisplay lightchain library.

Example 16 Adenoviral Expression Vector Construction

Using the Gateway® system, the antibody expression cassette of thepENABdisplay VH-VL library is recombined into the pAd/PL-DEST(Invitrogen, cat. V494-20) vector via the LR reaction following themanufacturer's instructions. The reaction is phenol-chloroformextracted, precipitated and transformed into the DH10B electrocompetentcells. Following plasmid DNA isolation of the resulting pAd/PL-VH-VLexpression vector, a digestion is performed with Pac I to expose leftand right viral ITRs and remove bacterial sequences. The ITR fragment isphenol-chloroform extracted, precipitated and transfected into HEK-293Acells using Lipofectamine 2000 (Invitrogen). The virus is harvestedafter the cytopathic effect is observed. Viral titers are determined byBD AdenoX Rapid titer kit (Becton Dickinson, CA).

pAd/PL-DEST is based on a replication incompetent adenovirus that canonly be propagated in cell lines that provide the E1 protein in trans(e.g., 293A cells). The pAd/PL-DEST based library described above iswell suited for screens using a cell line that conditionally expressesE1 protein, and thus conditionally supports viral replication. Such aline can be generated via stable transfection of suitable cells with anE1 protein expression construct comprising an inducible mammalianpromoter (e.g., tetracycline inducible promoters from clonetech). Thescreening process itself may be performed as describe in the examplebelow. E1 protein expression should be kept off during 1) infection withthe library, 2) incubation of infected cells to allow cell surfacedisplay of library encoded antibody 3) selection of the cells expressingan antibody with the desired characteristics. E1 protein expressionshould be induced in the selected cells comprising an adenovirusencoding an antibody with desired characteristics to promote viralreplication and thus to aid recovery of the virus.

Alternatively, the pENABdisplay heavy chain-light chain library may berecombined into a modified pAd/PL-DEST vector comprising the ts369mutation (Hasson, T. B. et al., J Virol 63(9):3612-21 (1989)). Methodsfor generating a pAd/PL-DEST vector comprising the ts369 mutation aredescribed bellow. Screen of library may be performed using a protocoldescribed in Example 17.

8.7 Example 17 Proof of Principle: Screen of an Artificial AdenoviralLibrary

pAd/PL-DEST vector comprising the ts369 mutation (Hasson, T. B. et al.,J Virol 63(9):3612-21 (1989)) is generated by replacing an RsrIIfragment (position 9666 to 17373) of the pAd/PL-DEST vector comprisingthe wild type sequence with a fragment comprising the ts369 mutation.Briefly, the RsrII fragment (position 9666 to 17373) of pAd/PL-DEST iscloned into a pUC18 vector with a modified multiple cloning site havingtwo RsrII sites. Basepair substitutions corresponding to ts369 areintroduced into the wild type fragment with the QuickChange kit(Stratagene) using oligonucleotides TS369F and TS369R (SEQ ID NO: 110and 111, respectively) according the manufacturer's instruction. TheRsrII fragment comprising ts369 is inserted into RsrII cut pAd/PL-DESTto generate pAd/PL-DEST/ts369. All of the cloning steps are performedusing standard laboratory protocols.

pENPABdisplay expression constructs comprising the 10C2 anti-EphA2antibody, the 3F2 anti-EphA2 antibody, the Abegrin anti-αvβ3 integrinantibody, an anti-PCDGF antibody, a 3F2 anti-EphA2 ScFvFc (single chainFv-Fc fusion), and a Abegrin anti-αvβ3 integrin ScFvFc are generatedusing standard cloning procedures. These expression constructs aredelivered into the pAd/PL-DEST/ts369 vector using the LR reaction of theGateway® (Invitrogen) system following the manufacturer'srecommendations. The LR reaction is phenol-chloroform extracted,precipitated and transformed into the DH10B electrocompetent cells.Following plasmid DNA isolation of the resulting pAd/PL/ts369 expressionvector, a digestion is performed with Pac I to expose left and rightviral ITRs and remove bacterial sequences. The ITR fragment isphenol-chloroform extracted, precipitated and transfected into HEK-293Acells using Lipofectamine 2000 (Invitrogen). The virus is harvestedafter the cytopathic effect is observed. Viral titers expressed as ViralParticle/ml (VP/ml) are determined using Quick Titer™ AdenovirusQuantitation Kit (Cell Biolabs, Inc). Multiplicity of infection (MOI) iscalculated based on VP/ml titer.

Artificial libraries were prepared by mixing aliquots of a) Abegrinanti-αvβ3 integrin antibody and 3F2 anti-EphA2 ScFvFc expressingviruses, b) an anti-PCDGF antibody and 10C2 anti-EphA2 antibodyexpressing viruses, and c) 3F2 anti-EphA2 antibody and Abegrin anti-αvβ3integrin ScFvFc expressing viruses to reach a final ratio of 100:1 viralparticles.

A polynucleotide encoding a human EphA2-Fc fusion protein consisting ofthe extracellular domain of human EphA2 fused with the Fc portion of ahuman IgG1 (SEQ ID NO: 112), can be generated via overlap PCR followingstandard protocols. Commercially available human cDNA may be used astemplate for the PCR reactions (e.g., FirstChoice® PCR-Ready andRACE-Ready cDNA from Ambion). Human EphA2-Fc fusion protein can beexpressed in human embryonic kidney (HEK) 293 cells and purified byprotein A affinity chromatography using standard protocols. HumanEphA2-Fc biotinylation may be carried out using an EZ-LinkSulfo-NHS-LC-Biotinylation Kit according to the manufacturer'sinstructions (Pierce, Rockford, Ill.).

Human αvβ3 integrin (Chemicon, #CC1018) is biotinylated utilizing anEZ-Link Sulfo-NHS-LC-Biotinylation Kit according to the manufacturer'sinstructions (Pierce, Rockford, Ill.).

Control experiments are performed to optimize the conditions for thedetection of cell surface displayed antibodies with αVβ3-biotin. 293Acells are infected at MOI=2.5 with adenoviruses encoding either theAbegrin anti-αvβ3 integrin ScFvFc or 3F2 anti-EphA2 ScFvFc. Infectedcells are incubated for 24 hrs at 40° C. Cells are harvested,resuspended at 4×10⁶cells/ml, and incubated in 4% milk at roomtemperature (RT) for 20 minutes. Separate aliquots of the infected cellsare contacted with 10 μg/ml, 5 μg/ml, 2.5 μg/ml, 1 μg/ml or 0.5 μg/mlαVβ3-biotin in 4% milk at RT for 30 minutes and on ice for an additional10 minutes. Cells are washed to remove any unbound sαVβ3-biotin andstained with FITC conjugated anti-human IgG-Fc (Pierce) orAPC-conjugated streptavidin (Pierce) following manufacturersrecommendations. Uninfected 293A cells are processed the same way andused as negative control. Fluorescently stained cells are analyzed on aflow cytometer. Data obtained are summarized in Table 10.

TABLE 10 Titration of αVβ3-biotin staining of Abegrin anti-αvβ3 integrinScFvFc (α- αVβ3) or 3F2 anti-EphA2 ScFvFc (α-EphA2) displaying cells.Percentage of positively stained cell sαVβ3-biotin 10 5 2.5 1 0.5 conc.used ug/ml ug/ml ug/ml ug/ml ug/ml secondary stain Fc^(a) Str^(b) Fc^(a)Str^(b) Fc^(a) Str^(b) Fc^(a) Str^(b) Fc^(a) Str^(b) surface Ab α-αVβ391 90 84 84 85 85 85 83 86 81 expressed α-EphA2 88 3 85 1 83 1 82 0 82 0none 2 0 3 0 1 0 0 0 0 0 ^(a)Fc secondary stain denotes FITC conjugatedanti-human IgG-Fc ^(b)Str secondary stain denotes APC-conjugatedstreptavidin

Proof of principle selection experiment I.: 293A cells (15×10⁶ cells) in30 ml of growth medium are plated in T-175 tissue culture flask the daybefore infection and incubated at 37° C. overnight. Cells are infectedwith the artificial library containing a mix of 3F2 anti-EphA2 antibodyand Abegrin anti-αvβ3 integrin ScFvFc expressing viruses at a ratio of100:1. Cells were infected at an MOI of 1-2.5 using standard protocols.Following 24 hrs of incubation at 40° C., the restrictive temperaturefor growth of ts369 mutant adenoviruses, infected cells are harvested,resuspended at 4×10⁶ cells/ml, and incubated in 4% milk at roomtemperature (RT) for 20 minutes. Cells are then contacted with 0.5-1μg/ml αVβ3-biotin in 4% milk at RT for 30 minutes and on ice for anadditional 10 minutes. Cells are washed to remove any unboundαVβ3-biotin. Cells with surface bound αVβ3-biotin are positivelyselected with a magnetic bead conjugated anti-biotin antibody (MiltenyiBiotech) following the manufacturer's instructions. Isolated cells aredouble stained with FITC conjugated anti-human IgG-Fc (Pierce) andAPC-conjugated streptavidin (Pierce) following manufacturersrecommendations. Stained cells are examined on a flow cytometer andcells displaying both FITC and APC staining are isolated using a FACSmachine. Flow cytometry profiles of cells representing various stages ofthe selection process, as well as the gate defining the selectioncriteria for sorting double positive cells are displayed in FIG. 13.Half of the isolated double positive cells are incubated at thepermissive temperature of 37° C. to allow for the recovery ofadenoviruses. Recovered virus may be subjected to a second round ofselection. The second half of the cells are lysed in lysis buffer (10 mMEDTA and 0.6% SDS), phenol-chloroform extracted, ethanol precipitated torecover viral DNA. Virus encoded antibody sequences are PCR amplifiedfrom the isolated DNA and cloned using standard procedures. Asufficiently large number of clones are sequenced to determine theefficiency of the selection process. 86% of the clones from theselection experiment depicted in FIG. 13 contained Abegrin anti-αvβ3integrin ScFvFc specific sequences; prior to selection Abegrin anti-αvβ3integrin ScFvFc represents 1% of the starting artificial library.

Proof of principle selection experiment II.: Artificial library usedcontains 100:1 ratio of viruses encoding Abegrin anti-αvβ3 integrinantibody and 3F2 anti-EphA2 ScFvFc. Biotinylated EphA2 ligand is usedfor detection of cell surface displayed 3F2 anti-EphA2 ScFvFc.Experiment was performed as described in paragraph [0250]. 34% of theclones derived from cells isolated in the selection experiment depictedin FIG. 14 contained 3F2 anti-EphA2 ScFvFc specific sequences; prior toselection 3F2 anti-EphA2 ScFvFc represents 1% of the starting artificiallibrary.

Proof of principle selection experiment III.: Artificial library usedcontains 100:1 ratio of viruses encoding an anti-PCDGF full lengthantibody and the 10C2 anti-EphA2 full length antibody. BiotinylatedEphA2 ligand is used for detection of cell surface displayed 10C2anti-EphA2 antibody. Experiment was performed as described. 87% of theclones derived from cells isolated in the selection experiment depictedin FIG. 15 contained 10C2 anti-EphA2 antibody specific sequences; priorto selection 10C2 anti-EphA2 antibody represents 1% of the startingartificial library.

Whereas, particular embodiments of the invention have been describedabove for purposes of description, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated by reference into thespecification to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated herein by reference.

1. A method for producing a library of cells displaying antibodies orfragments thereof on the cell surface comprising: a. introducing into apopulation of cells a library of vectors encoding recombinant antibodiesor fragments thereof that are displayed on the extracellular surface ofthe cell membrane; and b. culturing the population of cells underconditions that allow expression of the antibodies or fragments thereofon the cell surface, wherein the population of cells is a population ofmammalian cells and wherein said antibodies or fragments thereofcomprise the amino acid sequence of SEQ ID NO:
 60. 2. The method ofclaim 1, wherein the amino acid sequence of SEO ID NO: 60 is fused tothe C-terminal end of the heavy chain or the light chain of theantibody.
 3. The method of claim 1, wherein said antibodies or fragmentsthereof are of an immunoglobulin type selected from the group consistingof IgA, IgE, IgM, IgD, IgY and IgG.
 4. The method of claim 1, whereinsaid antibodies or fragments thereof are murine antibodies, chimericantibodies, humanized antibodies or human antibodies.
 5. The method ofclaim 1, wherein said antibodies or fragments thereof comprise an Fcregion, wherein the Fc region is a naturally occurring Fc region or avariant Fc region comprising at least one amino acid substitution,insertion, deletion or combination thereof.
 6. The method of claim 1,wherein said antibodies or fragments thereof comprise a heavy chainvariable region, a light chain variable region or both a heavy chain anda light chain variable region.
 7. The method of claim 1, wherein themammalian cells are selected from the group consisting of NS0 cells, CHOcells, Vero cells, COS7 cells, and 293 cells.
 8. The method of claim 1,wherein said vectors are viral vectors.
 9. The method of claim 8,wherein said vectors are adenoviral vectors, adeno associated viralvectors, herpes viral vectors or lentiviral vectors.
 10. A method ofdisplaying a library of antibodies or fragments thereof on the cellsurface comprising: a. introducing into a population of cells a libraryof vectors encoding recombinant antibodies or fragments thereof whereinsaid antibodies or fragments thereof comprise the amino acid sequence ofSEO ID NO: 60; and b. culturing the population of cells under conditionsthat allow expression of the antibodies or fragments thereof on the cellsurface, wherein the population of cells is a population of mammaliancells.
 11. The method of claim 10, wherein the mammalian cells areselected from the group consisting of NS0 cells, CHO cells, Vero cells,COS7 cells, and 293 cells.
 12. The method of claim 10, wherein saidantibody or fragment thereof is of an immunoglobulin type selected fromthe group consisting of IgA, IgE, IgM, IgD, IgY and IgG.
 13. The methodof claim 10, wherein said antibody or fragment thereof is a murineantibody, chimeric antibody, humanized antibody or human antibody. 14.The method of claim 10, wherein said antibody or fragment thereofcomprises an Fc region, wherein the Fc region is a naturally occurringFc region or a variant Fc region comprising at least one amino acidsubstitution, insertion, deletion or combination thereof.
 15. The methodof claim 10, wherein the amino acid sequence of SEQ ID NO: 60 is fusedto the C-terminal end of the heavy chain or the light chain of theantibody.